Use of the polypeptide compound

ABSTRACT

A pharmaceutical composition comprising an amount of a polypeptide of the formula:   &lt;IMAGE&gt;   wherein R1 is hydrogen or an acyl group, R2 is hydroxy or an acyloxy group, R3 is hydrogen, hydroxy or hydroxysulfonyloxy, R4 is hydrogen or carbamoyl, and R5 and R6 are each hydrogen or hydroxy, with the proviso that (i) R2 is acyloxy when R3 is hydrogen, and (ii) R5 is hydrogen when R6 is hydrogen, or a pharmaceutically acceptable salt thereof, is useful in the prevention or treatment of Pneumocystis carinii infection.

This application is a continuation of application Ser. No. 07/791,926,filed on Nov. 15, 1991, now abandoned, which is a continuation-in-partof application Ser. No. 07/614,125, filed Nov. 16, 1990, now abandoned,which is a continuation-in-part of application Ser. No. 07/610,759,filed Nov. 8, 1990, now abandoned.

The present invention relates to a new use of the polypeptide compoundor a pharmaceutically acceptable salt thereof.

More particularly, it relates to the utility of the polypeptide compoundfor the prevention and/or the treatment of Pneumocystis cariniiinfection (e.g., Pneumocystis carinii pneumonia, etc.) in a human beingor an animal.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide apharmaceutical composition for the prevention and/or the treatment ofPneumocystis carinii infection (e.g. Pneumocystis carinii pneumonia,etc.) in a human being or an animal comprising, as an active ingredient,the polypeptide compound or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for theprevention and/or the treatment of Pneumocystis carinii infection (e.g.Pneumocystis carinii pneumonia, etc.) in a human being or an animal,which comprises administering the polypeptide compound to a human beingor an animal.

A further object of the present invention is to provide a use of thepolypeptide compound for the manufacture of a medicament for theprevention and/or the treatment of Pneumocystis carinii infection (e.g.,Pneumocystis carinii pneumonia, etc.) in a human being or an animal.

The polypeptide compound used in the present invention is novel and canbe represented by the following general formula [I] (SEQ ID NO: 1):##STR2## wherein R¹ is hydrogen or an acyl group,

R² is hydroxy or an acyloxy group,

R³ is hydrogen, hydroxy or hydroxysulfonyloxy,

R⁴ is hydrogen or carbamoyl, and

R⁵ and R⁶ are each hydrogen or hydroxy, with proviso that

(i) R² is acyloxy when R³ is hydrogen, and

(ii) R⁵ is hydrogen when R⁶ is hydrogen.

The inventors of the present invention have found that the polypeptidecompound of the formula [I] and pharmaceutically acceptable saltsthereof are useful for the prevention and/or the treatment ofPneumocystis carinii infection (e.g., Pneumocystis carinii pneumonia,etc.) in a human being or an animal in need thereof. The polypeptidecompound of the formula [I] and pharmaceutically acceptable saltsthereof are also useful for the preparation of a medicament for theprevention and/or the treatment of Pneumocystis carinii infection (e.g.,Pneumocystis carinii pneumonia, etc.).

The pharmaceutical composition of the present invention can be used inthe form of a pharmaceutical preparation, for example, in solid,semisolid or liquid form, which contains the polypeptide compound [I] ora pharmaceutically acceptable salt thereof, as an active ingredient inadmixture with an organic or inorganic carrier or excipient suitable forrectal, pulmonary (nasal or buccal inhalation), nasal, ocular, external(topical), oral or parenteral (including subcutaneous, intravenous andintramuscular) administrations or insufflation.

The active ingredient may be compounded, for example, with the usualnon-toxic, pharmaceutically acceptable carriers for tablets, pellets,troches, capsules, suppositories, creams, ointments, aerosols, powdersfor insufflation, solutions, emulsions, suspensions, and any other formsuitable for use. If necessary or desired, additional auxiliary,stabilizing, thickening and coloring agents and perfumes may be used.

The polypeptide compound [I] and/or one or more pharmaceuticallyacceptable salt thereof is/are included in the pharmaceuticalcomposition in an amount sufficient to produce the desired effect uponthe process or condition of Pneumocystis carinii infection. Preferably,the amount of the polypeptide compound [I] and/or pharmaceuticallyacceptable salts thereof is sufficient either to effectively preventinfection by Pneumocystis carinii or to effectively treat a diseaseresulting from infection by Pneumocystis carinii.

The pharmaceutical composition of the present invention can bemanufactured by the conventional methods in this field of the art. Ifnecessary or desired, the techniques generally used in this field of theart for improving the bioavailability of a drug can be applied to thepharmaceutical composition of the present invention.

For administering the composition to a human being or an animal,preferable routes include intravenous, intramuscular, pulmonary, andoral administration, as well as insufflation.

While the dosage of therapeutically effective amount of the polypeptidecompound [I] varies from and also depends upon the age and condition ofeach individual patient to be treated, in the case of intravenousadministration, a daily dose of 0.01-100 mg of the polypeptide compound[I] per kg weight of a human being or an animal, preferably 0.1-50mg/kg, particularly preferably 0.5-30 mg/kg; in the case ofintramuscular administration, a daily dose of 0.1-100 mg of thepolypeptide compound [I] per kg weight of a human being or an animal,preferably 0.5-80 mg/kg, particularly preferably 1.0-50 mg/kg; and inthe case of oral administration, a daily dose of 0.5-100 mg of thepolypeptide compound [I] per kg weight of a human being or an animal,preferably 1.0-100 mg/kg, particularly preferably 2.0-75 mg/kg, isgenerally given for the prevention and/or the treatment of Pneumocystiscarinii infection (e.g., Pneumocystis carinii pneumonia, etc.) in ahuman being or an animal.

Especially preferred routes are described hereinbelow.

For administration by inhalation, the compounds of the present inventionare conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or nebulisers. The compounds may also bedelivered as powders which may be conventionally formulated, and thepowder compositions may be inhaled with the aid of an insufflationpowder inhaler device. The preferred delivery system for inhalation is ametered dose inhalation aerosol, which may be formulated as a suspensionor solution of compound in one or more suitable propellants, such asfluorocarbons or hydrocarbons.

Because of the desirability to directly treat lung and bronchi, aerosoladministration is a preferred method of administration. Insufflation isalso a desirable method, especially where infection may have spread toears ahd other body cavities.

Alternatively, parenteral administration may be employed using dripintravenous administration.

In order to show the usefulness of the polypeptide compound [I] or apharmaceutically acceptable salt thereof used in the present inventionfor the prevention and/or the treatment of Pneumocystis cariniiinfection (e.g., Pneumocystis carinii pneumonia, etc.) in a human beingor an animal, the pharmacological test data of the representativecompounds thereof are shown in the following tests.

Test 1

Eight Hooded nude rats (3 male, 5 female) were intranasally infectedwith 10⁴ Pneumocystis carinii cysts (derived from rat), andsubcutaneously injected with 20 mg cortisone once a week for 8 weeks. Atthe start of the treatment with FR131535 substance, three of the 8 ratswere sacrificed. The lungs were removed, homogenized with a glasshomogenizer in phosphate buffered saline, and processed forquantification as described below. The remaining rats were divided intotwo groups, and the rats of one group were intraperitoneally injectedonce daily with 2 mg of FR131535 substance in 0.5 ml of saline and therats of the other group were intraperitoneally injected once daily onlywith 0.5 ml of saline as a negative control.

The total number of cysts per rat lung was determined by quantifying thenumber of cysts of homogenized lung tissue on slides fixed withether/sulfuric acid and stained with toluidine blue 0.

The test results were as follows.

    ______________________________________                                                                  Number of cysts                                     Treatment with     Rat    per lung (log.sub.10)                               ______________________________________                                        before                 1      6.56                                            treatment              2      6.53                                                                   3      6.30                                            FR131535   10 mg.sup.a (5.sup.b)                                                                     4      4.56                                            substance  12 mg.sup.a (6.sup.b)                                                                     5      3.89                                                       26 mg.sup.a (13.sup.b)                                                                    6      4.78                                            saline                 7      7.15                                                                   8      7.94                                            ______________________________________                                         .sup.a Total amount of FR131535 substance given to rat                        .sup.b the day after the start of treatment when the number of cysts in       lung was determined                                                      

Test 2

1. Test Method

Sixty-three female BALB/C nu/nu mice, 5 weeks old, were intranasallyinfected with 10⁴ cysts per head under anesthesia, housed in sterilizedvinyl isolators and fed under completely sterilized conditions.

Experiment I: Prophylactic effect

Fifty-two days after the infection (at this time, Pneumocystis cariniipneumonia had not yet occurred, judging from the conditions of themice), 34 of the infected mice were randomly selected and used for theprophylactic experiment.

Five mice were sacrificed for determining the physical condition at thestarting point.

One of the lungs from each mouse was removed, weighed, and preserved at-80° C. for examining the number of cysts in the lung. The number ofcysts was determined by microscopically counting the number of cysts ofhomogenized lung tissue on a slide fixed with ether/sulfuric acid andstained with toluidine blue 0 (hereinafter, this procedure is referredto as "Protocol").

The remaining 29 mice were divided into three groups. Nine mice(Group 1) were subcutaneously injected with saline once a day except onSaturday and Sunday. Two groups of 10 mice were similarly treated with10 mg/kg of FR 131535 substance (Group 2) and FR 901379 substance (Group3), respectively.

Half of the mice of each group were sacrificed 18 days after the startof the administration (Point A) and examined by the "Protocol", and theremainder were similarly examined 56 days after the start of theadministration (Point B).

Experiment II: Curative effect

The mice not used in Experiment I began to show typical symptoms ofPneumocystis carinii pneumonia, mainly wasting and cyanosis, about threemonths after the infection.

One hundred one days after the infection, the mice were divided into twogroups, based on the degree of the symptoms (light symptoms: 17 mice,heavy symptoms: 14 mice). Each group was divided into four subgroups.

One subgroup (5 mice) out of each group of four subgroups was sacrificedfor determining the status of mice at the start of therapy by the"Protocol".

Administration of the drugs was carried out in the same manner asExperiment I once a day except on Saturday and Sunday [heavy symptomsgroup: 3 mice for saline (Group 4), 3 mice for FR 131535 substance(Group 5) and 3 mice for FR 901379 substance (Group 6); light symptomsgroup: 4 mice for saline (Group 7), 4 mice for FR 131535 substance(Group 8) and 4 mice for FR 901379 substance (Group 9)).

The mice were treated for two weeks, then sacrificed at the end of thetherapy for the examination of the number of the total cysts in the samemanner as in the "Protocol".

2. Test Results

    ______________________________________                                        Test Group        Total Cysts (mean log.sub.10)                               ______________________________________                                        Experiment I                                                                  at Point A                                                                    Group 1           5.35 ± 0.26                                              Group 2           1.76 ± 0.03***                                           Group 3           1.81 ± 0.06***                                           at Point B                                                                    Group 1           6.34 ± 0.14                                              Group 2           2.31 ± 0.26***                                           Group 3           2.04 ± 0.25***                                           Experiment II                                                                 heavy symptoms                                                                Group 4           6.20 ± 0.08                                              Group 5           2.06 ± 0.06***                                           Group 6           3.10 ± 0.513***                                          light symptoms                                                                Group 7           6.21 ± 0.12                                              Group 8           3.85 ± 0.21***                                           Group 9           3.23 ± 0.67***                                           ______________________________________                                         ***P<0.001                                                               

From the above test results, it turned out that the polypeptide compound[I] or a pharmaceutically acceptable salt thereof used in the presentinvention was very useful for the prevention and/or the treatment ofPneumocystis carinii pneumonia.

In the following, the polypeptide compound [I] or a pharmaceuticallyacceptable salt thereof used in the present invention is explained indetail.

The polypeptide compound or a salt thereof can be prepared by theprocesses as illustrated in the following schemes. ##STR3## wherein R³,R⁴, R⁵ and R⁶ are each defined above,

R_(a) ¹ is an acyl group,

R_(b) ¹ is an ar(lower)alkanoyl which has one or more higher alkoxygroup(s) and a protected amino group,

R_(c) ¹ is an ar(lower)alkanoyl which has one or more higher alkoxygroup(s) and an amino group,

R_(d) ¹ is halo(lower)alkanoyl

R_(e) ¹ is pyridylthio(lower)alkanoyl which may have one or more higheralkyl group(s),

R_(f) ¹ is acyloxy,

R_(a) ¹ is an acyl group,

R⁷ is an acyl group, and

R_(a) ³ is hydroxy or hydroxysulfonyloxy

Some of the starting compounds [III] (SEQ ID NO: 1) are novel and can beprepared according to the aforesaid Processes 3 to 6.

Suitable pharmaceutically acceptable salts of the object compound [I]are conventional non-toxic mono- or di-salts, and include metal saltssuch as alkali metal salts [e.g., sodium salt, potassium salt, etc.],alkaline earth metal salts [e.g., calcium salt, magnesium salt, etc.],ammonium salt(s), organic base salts [e.g., trimethylammonium salt,triethylammonium salt, pyridinium salt, picolinium salt,dicyclohexylammonium salt, N,N-dibenzylethylenediammonium salt, etc.],organic acid addition salts [e.g., formate, acetate, trifluroacetate,maleate, tartrate, methanesulfonate, benzenesulfonate, and/ortoluenesulfonate salts, etc.], inorganic acid addition salts [e.g.,hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.], asalt with one or more amino acids [e.g., arginine salt, aspartic acidsalt, glutamic acid salt, etc.], and the like.

In the above and subsequent description of this specification, suitableexamples of the various definitions are explained in detail as follows:

The term "lower" is intended to mean 1 to 6 carbon atom(s), unlessotherwise indicated.

The term "higher" is intended to mean 7 to 20 carbon atoms, unlessotherwise indicated.

A suitable "acyl group" may be an aliphatic acyl, aromatic acyl,heterocyclic acyl, arylaliphatic acyl and heterocyclic-aliphatic acylderived from a carboxylic acid, carbonic acid, carbamic acid, sulfonicacid, and the like.

Suitable example of the "acyl group" thus explained include:

lower alkanoyl [e.g., formyl, acetyl, propionyl, butyryl, isobutyryl,valeryl, hexanoyl, pivaloyl, etc.] which may have one or more(preferably 1 to 3) suitable substituent(s) such as halogen (e.g.,fluoro, chloro, bromo, iodo); aryl (e.g. phenyl, naphthyl, anthryl,etc.) which may have one or more (preferably 1 to 3) suitablesubstituent(s) like hydroxy, higher alkoxy as explained below, aforesaidaryl, or the like; lower alkoxy as explained below; amino; protectedamino, preferably acylamino, such as lower alkoxycarbonylamino (e.g.,methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino,butoxycarbonylamino, t-butoxycarbonylamino, pentyloxycarbonylamino,hexyloxycarbonylamino, etc.), or the like; di(lower)alkylamino (e.g.,dimethylamino, N-methylethylamino, diethylamino, N-propylbutylamino,dipentylamino, dihexylamino, etc.); lower alkoxyimino (e.g.,methoxyimino, ethoxyimino, propoxyimino, butoxyimino, t-butoxyimino,pentyloxyimino, hexyloxyimino, etc.); ar(lower)alkoxyimino such asphenyl(lower)alkoxyimino (e.g., benzyloxyimino, phenethyloxyimino,benzhydryloxyimino, etc.) which may have one or more (preferably 1 to 3)suitable substituent(s) like higher alkoxy as explained below, or thelike; heterocyclicthio, preferably pyridylthio, which may have one ormore (preferably 1 to 3) suitable substituent(s) like higher alkyl(e.g., heptyl, octyl, 2-ethylhexyl, nonyl, decyl, 3,7-dimethyloctyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,3-methyl-10-ethyldodecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,eicosyl, etc.), or the like; heterocyclic group (e.g., thienyl,imidazolyl, pyrazolyl, furyl, tetrazolyl, thiazolyl, thiadiazolyl, etc.)which may have one or more (preferably 1 to 3) suitable substituent(s)like amino, aforesaid protected amino, aforesaid higher alkyl, or thelike; or the like;

higher alkanoyl [e.g., heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl,10,12-dimethyltetradecanoyl, heptadecanoyl, stearoyl, nonadecanoyl,eicosanoyl, etc.];

lower alkenoyl [e.g., acryloyl, methacryloyl, crotonoyl, 3-pentenoyl,5-hexenoyl, etc.] which may have one or more (preferably 1 to 3)suitable substituent(s) such as aforesaid aryl which may have one ormore (preferably 1 to 3) suitable substituent(s) like higher alkoxy asexplained below, or the like; or the like;

higher alkenoyl [e.g., 4-heptenoyl, 3-octenoyl, 3,6-decadienoyl,3,7,11-trimethyl-2,6,10-dodecatrienoyl, 4,10-heptadecadienoyl, etc.];

lower alkoxycarbonyl [e.g., methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl,hexyloxycarbonyl, etc.];

higher alkoxycarbonyl [e.g., heptyloxycarbonyl, octyloxycarbonyl,2-ethylhexyloxycarbonyl, nonyloxycarbonyl, decyloxycarbonyl,3,7-dimethyloctyloxycarbonyl, undecyloxycarbonyl, dodecyloxycarbonyl,tridecyloxycarbonyl, tetradecyloxycarbonyl, pentadecyloxycarbonyl,3-methyl-10-ethyldodecyloxycarbonyl, hexadecyloxycarbonyl,heptadecyloxycarbonyl, octadecyloxycarbonyl, nonadecyloxycarbonyl,eicosyloxycarbonyl, etc.];

aryloxycarbonyl [e.g., phenoxycarbonyl, naphthyloxycarbonyl, etc.];

arylglyoxyloyl [e.g., phenylglyoxyloyl, naphthylglyoxyloyl, etc.];

ar(lower)alkoxycarbonyl which may have one or more suitablesubstituent(s) such as phenyl(lower)alkoxycarbonyl which may have anitro or lower alkoxy group [e.g., benzyloxycarbonyl,phenethyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, etc.];

lower alkylsulfonyl [e.g., methylsulfonyl, ethylsulfonyl,propylsulfonyl, isopropylsulfonyl, pentylsulfonyl, butylsulfonyl, etc.];

arylsulfonyl [e.g., phenylsulfonyl, naphthylsulfonyl etc.] which mayhave one or more (preferably 1 to 3) suitable substituent(s) such aslower alkyl as explained below, higher alkoxy as explained below, or thelike;

ar(lower)alkylsulfony such as phenyl(lower)alkylsulfonyl [e.g.,benzylsulfonyl, phenethylsulfony, benzhydrylsulfonyl, etc.], or thelike; and

aroyl [e.g., benzoyl, naphthoyl, anthrylcarbonyl, etc.] which may haveone or more (preferably 1 to 5) suitable substituent(s) such asaforesaid halogen; lower alkyl (e.g., methyl, ethyl, propyl, butyl,t-butyl, pentyl, hexyl, etc.); aforesaid higher alkyl; lower alkoxy(e.g., methoxy, ethoxy, propoxy, butoxy, t-butoxy, pentyloxy hexyloxy,etc.) which may have one or more (preferably 1 to 10) suitablesubstituent(s) like aforesaid lower alkoxy, aforesaid halogen, aforesaidaryl, or the like; higher alkoxy (e.g., heptyloxy, octyloxy,2-ethylhexyloxy, nonyloxy, decyloxy, 3,7-dimethyloctyloxy, undecyloxy,dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy,3-methyl-10-ethyldodecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy,nonadecyloxy, eicosyloxy, etc.) which may have one or more (preferably 1to 17) suitable substituent(s) like aforesaid halogen; higher alkenyloxy(e.g., 3-heptenyloxy, 7-octenyloxy, 2,6-octadienyloxy, 5-nonenyloxy,1-decenyloxy, 3,7-dimethyl-6-octenyloxy, 3,7-dimethyl-2,6-octadienyloxy,8-undecenyloxy, 3,6,8-dodecatrienyloxy, 5-tridecenyloxy,7-tetradecenyloxy, 1,8-pentadecadienyloxy, 15-hexadecenyloxy,11-heptadecenyloxy, 7-octadecenyloxy, 10-nonadecenyloxy,18-eicosenyloxy, etc.); carboxy; aforesaid aryl which may have one ormore (preferably 1 to 3) suitable substituent(s) like aforesaid higheralkoxy; aryloxy (e.g. phenoxy, naphthyloxy, anthryloxy, etc.) which mayhave one or more (preferably 1 to 3) suitable substituent(s) likeaforesaid lower alkoxy, or aforesaid higher alkoxy; or the like; or thelike.

Preferred "acyl groups" include:

lower alkanoyl or halo(lower)alkanoyl;

ar(lower)alkanoyl which may have one or more (preferably 1 to 3)suitable substituent(s) such as hydroxy, lower alkoxy, higher alkoxy,aryl, amino, protected amino, di(lower)alkylamino, lower alkoxyimino orar(lower)alkoxyimino which may have one or more (preferably 1 to 3)higher alkoxy group(s);

heterocyclicthio(lower)alkanoyl which may have one or more (preferably 1to 3) higher alkyl group(s);

heterocyclic(lower)alkanoyl which may have one or more (preferably 1 to3) suitable substituent(s) such as lower alkoxyimino, higher alkyl,amino or protected amino;

ar(lower)alkoxyimino(lower)alkanoyl which may have one or more(preferably 1 to 3) higher alkoxy group(s);

higher alkanoyl;

ar(lower)alkenoyl which may have one or more (preferably 1 to 3) higheralkoxy group(s);

higher alkenoyl; lower alkoxycarbonyl; higher alkoxycarbonyl;aryloxycarbonyl;

arylsulfonyl which may have one or more (preferably 1 to 3)substituent(s) such as lower alkyl or higher alkoxy; and

aroyl which may have one or more (preferably 1 to 5) suitablesubstituent(s) such as halogen, lower alkyl, higher alkyl, carboxy,lower alkoxy which may have one or more (preferaby 1 to 10) halogenatom(s), lower alkoxy(lower)alkoxy, ar(lower)alkoxy, higher alkoxy whichmay have one or more (preferably 1 to 17) halogen atom(s), higheralkenyloxy, aryl which may have one or more (preferably 1 to 3) higheralkoxy group(s) or aryloxy which may have one or more (preferably 1 to3) substituent(s) such as lower alkoxy or higher alkoxy.

The more preferred acyl group include lower alkanoyl;halo(lower)alkanoyl;

phenyl(lower)alkanoyl or naphthyl(lower)alkanoyl, each of which may have1 to 3 substituents such as hydroxy, lower alkoxy, higher alkoxy,phenyl, amino, lower alkoxycarbonylamino, di(lower)alkylamino, loweralkoxyimino, or phenyl(lower)alkoxyimino which may have 1 to 3 higheralkoxy group(s);

pyridylthio(lower)alkanoyl which may have 1 to 3 higher alkyl group(s);

imidazolyl(lower)alkanoyl or thiazolyl(lower)alkanoyl, each of which mayhave 1 to 3 suitable substituents such as lower alkoxyimino, higheralkyl, amino or lower alkoxycarbonylamino;

phenyl(lower)alkoxyimino(lower)alkanoyl which may have 1 to 3 higheralkoxy group(s);

higher alkanoyl;

phenyl(lower)alkenoyl which may have 1 to 3 higher alkoxy group(s);

higher alkenoyl; lower alkoxycarbonyl, higher alkoxycarbonyl;phenoxycarbonyl;

phenylsulfonyl or naphthylsulfonyl, each of which may have 1 to 3 loweralkyl or higher alkoxy group(s); and

benzoyl, naphthoyl or anthrylcarbonyl, each of which may have 1 to 5suitable substituents such as halogen, lower alkyl, higher alkyl,carboxy, lower alkoxy which may have 6 to 10 halogen atoms, loweralkoxy(lower)alkoxy, phenyl(lower)alkoxy, higher alkoxy which may have12 to 17 halogen atom(s), higher alkenyloxy, phenyl which may have 1 to3 higher alkoxy group(s), phenoxy which may have 1 to 3 lower alkoxy orhigher alkoxy group(s).

The much more preferred acyl groups include:

(C₁ -C₄)alkanoyl; halo(C₁ -C₄)alkanoyl;

phenyl(C₁ -C₄)alkanoyl which may have 1 to 3 suitable substituent (s)such as hydroxy, (C₁ -C₄)alkoxy, (C₇ -C₁₆)alkoxy, phenyl, amino, (C₁-C₄)alkoxycarbonylamino, di(C₁ -C₄)alkylamino, (C₁ -C₄)alkoxyimino orphenyl(C₁ -C₄)alkoxyimino which may have a (C₇ -C₁₆)alkoxy group;

naphthyl(C₁ -C₄)alkanoyl which may have 1 to 3 (C₁-C₄)alkoxycarbonylamino groups;

1-(C₇ -C₁₆)alkylpyridiniothio(C₁ -C₄)alkanoyl;

imidazolyl(C₁ -C₄)alkanoyl which may have 1 to 3 (C₇ -C₁₆)alkyl or (C₁-C₄)alkoxycarbonylamino group(s);

thiazolyl(C₁ -C₄)alkanoyl which may have 1 to 3 (C₁ -C₄)alkoxyimino oramino group(s);

phenyl (C₁ -C₄)alkoxyimino(C₁ -C₄) alkanoyl which may have 1 to 3 (C₇-C₁₆)alkoxy group(s);

(C₇ -C₁₇)alkyl;

phenyl(C₁ -C₄)alkenoyl which may have 1 to 3 (C₇ -C₁₆)alkoxy group(s);

(C₇ -C₁₈)alkenoyl; (C₃ -C₆)alkoxycarbonyl;

(C₇ -C₁₆)alkoxycarbonyl; phenoxycarbonyl;

phenylsulfonyl which may have a (C₁ -C₄)alkyl or (C₇ -C₁₆)alkoxy group;

naphthylsulfonyl which may have a (C₇ -C₁₆)alkoxy group;

benzoyl which may have 1 to 5 suitable substituent(s) such as halogen,(C₃ -C₆)alkyl, (C₇ -C₁₆)alkyl, carboxy, (C₁ -C₆)alkoxy which may have 6to 10 halogen atoms, (C₁ -C₄)alkoxy(C₁ -C₄)alkoxy, phenyl(C₃ -C₆)alkoxy,(C₇ -C₁₆)alkoxy which may have 12 to 17 halogen atoms, phenyl which mayhave 1 to 3 (C₇ -C₁₆)alkoxy group(s) or phenoxy which may have 1 to 3(C₃ -C₆)alkoxy or (C₇ -C₁₆)alkoxy group(s);

naphthoyl which may have 1 to 3 suitable substituent(s) such as (C₃-C₆)alkoxy, (C₇ -C₁₆)alkoxy or (C₇ -C₁₆)alkenyloxy; and

anthrylcarbonyl.

The most preferred acyl groups are acetyl, 2-bromoacetyl,2-(4-biphenylyl)acetyl, 2-(4-octyloxyphenyl)acetyl,3-(4-octyloxyphenyl)propionyl, 2-amino-2-(4-octyloxyphenyl)acetyl,2-(t-butoxycarbonylamino)-2-(4-octyloxyphenyl)acetyl,2-amino-3-(4-octyloxyphenyl)propionyl,2-(t-butoxycarbonylamino)-3-(4-octyloxyphenyl)propionyl,2-dimethylamino-3-(4-octyloxyphenyl)propionyl,2-(t-butoxycarbonylamino)-2-(2-naphthyl)acetyl,2-methoxy-2-(4-octyloxyphenyl)acetyl,2-methoxyimino-2-(4-octyloxyphenyl)acetyl,2-(4-octyloxybenzyloxyimino)-2-(4-hydroxyphenyl)acetyl,2-(4-octyloxybenzyloxyimino)-2-phenylacetyl,2-(4-octyloxybenzyloxyimino)acetyl, 2-(1-octyl-4-pyridinio)thioacetyl,2-methoxyimino-2-(2-aminothiazol-4-yl)acetyl,2-(t-butoxycarbonylamino)-3-(1-octyl-4-imidazolyl)propionyl,3-(4-octyloxyphenyl)acryloyl, 3,7,11-trimethyl-2,6,10-dodecatrienoyl,t-butoxycarbonyl, octyloxycarbonyl, phenoxycarbonyl, p-tolylsulfonyl,4-octyloxyphenylsulfonyl, 6-octyloxy-2-naphthylsulfonyl,4-(t-butyl)benzoyl, 4-octylbenzoyl,2,3,5,6-tetrafluoro-4-(2,2,3,3,4,4,5,5-octafluoropentyloxy)benzoyl,4-(2-butoxyethoxy)benzoyl, 4-(4-phenylbutoxy)benzoyl, 4-octyloxybenzoyl,2-carboxy-4-octyloxybenzoyl, 3-methoxy-4-octyloxybenzoyl,4-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyloxy)-2,3,5,6-tetrafluorobenzoyl,4-(4-octyloxyphenyl)benzoyl, 4-(4-octyloxyphenoxy)benzoyl,6-butoxy-2-naphthoyl, 6-hexyloxy-2-naphthoyl, 6-octyloxy-2-naphthoyl,6-(2-ethylhexyloxy)-2-naphthoyl, 6-decyloxy-2-naphthoyl,6-(3,7-dimethyloctyloxy)-2-naphthoyl, 6-dodecyloxy-2-naphthoyl,6-(3,7-dimethyl-6-octenyloxy)-2-naphthoyl,6-(3,7-dimethyl-2,6-octadienyloxy)-2-naphthoyl, 2-anthrylcarbonyl,4-(4-heptyloxyphenyl)benzoyl and 4-(4-hexyloxyphenoxy)benzoyl.

Suitable "ar(lower)alkanoyl" moieties in the groups "ar(lower)alkanoylwhich has higher alkoxy and protected amino" and "ar(lower)alkanoylwhich has higher alkoxy and amino" are selected from those describedabove for "acyl group", and suitable examples of the substituent(s)"higher alkoxy" and "protected amino" are also selected from thosedescribed above for "acyl group".

Suitable "halo(lower)alkanoyl" are selected from those described abovefor "acyl group".

Suitable "pyridylthio(lower)alkanoyl" in "pyridylthio(lower)alkanoylwhich may have higher alkyl" are chosen from the ones as exemplifiedabove for "acyl group", and suitable examples of the substituent "higheralkyl" are chosen from those exemplified above for "acyl group".

Suitable "acyloxy" may include hydroxysulfonyloxy, phosphonooxy, and thelike.

In the object compound [I] thus defined, the following compound [Ik](SEQ ID NO: 1) is especially preferable: ##STR4## wherein R¹ is hydrogenor an acyl group.

A suitable "acylating agent" for the acylation reaction in Process 4 maybe an acid compound corresponding to the acyl group to be introduced, ora salt thereof, or its reactive derivative at the carbon group. Asuitable example of said acylating agent is represented by the formula:

    R.sub.a.sup.1 --OH                                         [V]

wherein R_(a) ¹ is as defined above, or a salt thereof, or its reactivederivative at the carboxy group.

Suitable reactive derivatives of the "acylating agent" above includeacid halides, such as an acid chloride, acid anhydrides, which may besymmetrical or unsymmetrical, or may be another known reactivederivative. The phrase "reactive derivative at the carboxy group" meansa reactive moiety in place of the --OH group in formula [IV] above,which will facilitate the reaction of acylating agent with the primaryamino group of the compound of formula [Id].

In the compound [V], the following compounds are novel: ##STR5## or itsreactive derivative at the carboxy group,

or a salt thereof, ##STR6## or its reactive derivative at the carboxygroup,

or a salt thereof,

wherein

R⁸ is lower alkoxy, higher alkoxy or higher alkenyloxy,

R⁹ is --COOH or --SO₃ H,

R₁₀ is hydrogen or halogen,

R₁₁ is lower alkoxy which has one or more halogen atoms, or higheralkoxy which has one or more halogen atoms, wherein the maximum numberof halogen atoms is represented by a perhalogenated substituent.

The compounds [V-1] and [V-2] can be prepared by the followingprocesses: ##STR7## wherein R⁸, R⁹, R¹⁰ and R¹¹ are each as definedabove,

R¹² is lower alkyl, higher alkyl or higher alkenyl,

R¹³ is lower alkyl which has one or more halogen atom(s) or higher alkylwhich has one or more halogen atom(s) , and

X and Y are each a leaving group.

In the above definitions, suitable "lower alkoxy", "higheralkoxy""higher alkenyloxy", "halogen", "lower alkyl" and "higher alkyl"are defined as described and exemplified above.

Suitable "higher alkenyl" groups may include 3-heptenyl, 7-octenyl,2,6-octadienyl, 5-nonenyl, 1-decenyl, 3,7-dimethyl-6-octenyl,3,7-dimethyl-2,6-octadienyl, 8-undecenyl, 3,6,8-dodecatrienyl,5-tridecenyl, 7-tetradecenyl, 1,8-pentadecadienyl, 15-hexadecenyl,11-heptadecenyl, 7-octadecenyl, 10-nonadecenyl, 18-eicosenyl and thelike. The preferred higher alkenyl group is a (C₇ -C₁₆)alkenyl group.

As for R¹¹, "lower alkoxy" has one or more (preferably 1 to 10, morepreferably 6 to 10) halogen atom(s), and "higher alkoxy" has one or more(preferably 1 to 17, more preferably 12 to 17) halogen atom(s).

As for R¹³, "lower alkyl" has one or more (preferably 1 to 10, morepreferably 6 to 10) halogen atom(s), and "higher alkyl" has one or more(preferably 1 to 17, more preferably 12 to 17) halogen atom(s).

As for R⁸, the preferred "lower alkoxy" is (C₄ -C₆)alkoxy.

A suitable "leaving group" includes aforesaid halogen, lower alkanoyloxy(e.g., acetoxy, etc.), sulfonyloxy (e.g., mesyloxy, toxyloxy, etc.), andthe like.

Suitable salts and reactive derivatives at the carboxy group of thecompounds [V-1] and [V-2] include those described above and the onesexemplified below for the compound [V].

The reactions in Processes A and B can be carried out according to themethods disclosed later in Preparations of the present specification, orknown methods similar thereto.

For the compounds of the formula [V], there are other novel compoundsthan those of the formulae [V-1] and [V-2], and they can be prepared,for example, by the methods disclosed later in Preparations.

A suitable "pyridinethione" in Process 6 may include1,2-dihydropyridine-2-thione, 1,4-dihydropyridine-4-thione, or the like,and said "pyridinethione" may have a "higher alkyl" group as describedabove.

The processes for preparing the object compound [I] of the presentinvention or a salt thereof are explained in detail in the followingProcess and Preparations.

Process 1

The object compound [Ia] (SEQ ID NO: 1) or a salt thereof can beprepared by a fermentation process.

The fermentation process is explained in detail hereinbelow.

The compound [Ia] of the present invention or a salt thereof can beproduced by fermentation of a strain belonging to the genus Coleophoma,such as Coleophoma sp. F-11899, which produces the compound [Ia] or asalt thereof in a nutrient medium.

(i) Microorganism:

Particulars of the microorganism used for producing the compound [Ia] ora salt thereof is explained hereinbelow.

The strain F-11899 was originally isolated from a soil sample collectedat Iwaki-shi, Fukushima-ken, Japan. This organism grew ratherrestrictedly on various culture media, and formed dark grey to brownishgrey colonies. An anamorph (conidiomata) was produced on asteam-sterilized leaf segment affixed on a Miura's LCA plate (Miura, K.and Kudo, M. Y.: An agar-medium for aquatic Hyphomycetes. Trans. Ycolo.Soc. Japan, 11:116-118, 1970), and on a corn meal agar plate byinoculating the isolate, while neither a teleomorph nor an anamorphformed on agar media alone. Its morphological, cultural andphysiological characteristics are as follows.

Cultural characteristics on various agar media are summarized inTable 1. Cultures on potato dextrose agar grew rather rapidly, attaining3.5-4.0 cm in diameter after two weeks at 25° C. This colony surface wasplanar, felty, somewhat wrinkly and brownish grey. The colony center waspale grey to brownish grey, and covered with aerial hyphae. The reversecolor was dark grey. Colonies on malt extract agar grew morerestrictedly, attaining 2.5-3.0 cm in diameter under the sameconditions. The surface was planar, thin to felty and olive brown. Thecolony center was yellowish grey, and covered with aerial hyphae. Thereverse was brownish grey.

The morphological characteristics were determined on basis of thecultures on a sterilized leaf affixed to a Miura's LCA plate.Conidiomata formed on the leaf segment alone. They were pycnidial,superficial, separate, discoid to ampulliform, flattened at the base,unilocular, thin-walled, black, 90-160(-200) μm in diameter and 40-70 μmhigh. Ostiole was often single, circular, central, papillate, 10-30 μmin diameter and 10-20 μm high. Conidiophores formed from the lower layerof inner pycnidial walls. They were hyaline, simple or sparinglybranched, septate and smooth. Conidiogenous cells were enteroblastic,phialidic, determinate, ampulliform to obpyriform, hyaline, smooth, 5-8x 4-6 μm, with a collarette. The collarettes were campanulate tocylindrical, and 14-18×3-5 μm. Conidia were hyaline, cylindrical,thin-walled, aseptate, smooth and 14-16(-18)×2-3 μm.

The vegetative hyphae were septate, brown, smooth and branched. Thehyphal cells were cylindrical and 2-7 μm thick. The chlamydospores wereabsent.

The strain F-11899 had a temperature range for growth of 0°to 31° C. andan optimum temperature of 23° to 27° C. an potato dextrose agar.

The above characteristics indicate that the strain F-11899 belongs tothe order Coelomycetes (von Arx, J. A.: "The Genera ofFungi--Sporulating in Pure Culture," 3rd ed ., J. Cramer, ed., Vaduz,1974; Sutton, B. C.: "The Coelomycetes--Fungi Imperfecti with Pycnidia,Acervuli and Stromata," Commonwealth Mycological Institute, Kew, 1980;Hawksworth, D. L., Sutton, B. C., and Ainsworth, G. C.: "Dictionary ofthe Fungi," 7th ed. Commonwealth Mycological Institute, Kew, 1983).Thus, we named the strain "Coelomycetes strain F-11899".

                  TABLE 1                                                         ______________________________________                                        Cultural characteristics of the strain F-11899                                Medium       Cultural characteristics                                         ______________________________________                                        Malt extract agar                                                                          G:    Rather restrictedly, 2.5-3.0 cm                            (Blakeslee 1915)                                                                           S:    Circular, planar, thin to felty,                                              olive brown (4F5), arising aerial                                             hyphae at the center (yellowish grey                                          (4B2))                                                                  R:    Brownish grey (4F2)                                        Potato dextrose agar                                                                       G:    Rather rapidly, 3.5-4.0 cm                                 (Difco 0013) S:    Circular, planar, felty, somewhat                                             wrinkly, brownish grey (4F2),                                                 arising aerial hyphae at the center                                           (pale grey (4B1) to brownish grey                                             (4F2))                                                                  R:    Dark grey (4F1)                                            Czapeck's solution                                                                         G:    Very restrictedly, 1.0-1.5 cm                              agar (Raper and                                                                            S:    Irregular, thin, scanty, immersed,                         Thom 1949)         subhyaline to white                                                     R:    Subhyaline to white                                        Sabouraud dextrose                                                                         G:    Restrictedly, 2.0-2.5 cm                                   agar (Difco 0109)                                                                          S:    Circular, planar, thin, white,                                                sectoring, light brown (6D5) at the                                           colony center                                                           R:    Pale yellow (4A3)                                          Oatmeal agar G:    Fairly rapidly, 4.0-4.5 cm                                 (Difco 0552) S:    Circular, planar, felty to cottony,                                           dark grey (4F1) to brownish grey                                              (4F2)                                                                   R:    Brownish grey (4D2)                                        Emerson Yp Ss agar                                                                         G:    Restrictedly, 2.0-2.5 cm                                   (Difco 0739) S:    Circular to irregular, planar,                                                felty, dark grey (4F1) to brownish                                            grey (4F2)                                                              R:    Medium grey (4E1) to dark grey                                                (4F1)                                                      Corn meal agar                                                                             G:    Rather restrictedly, 2.5-3.0 cm                            (Difco 0386) S:    Circular, planar, thin to felty,                                              dark grey (2F1) to olive (2F3)                                          R:    Dark grey (2F1) to olive (2F3)                             MY20 agar    G:    Restrictedly, 1.5-2.0 cm                                                S:    Circular to irregular, thin,                                                  sectoring, yellowish white (4A2)                                        R:    Pale yellow (4A3) to orange white                                             (5A2)                                                      Abbreviations:                                                                             G:    growth, measuring colony size in                                              diameter                                                                S:    colony surface                                                          R:    reverse                                                    ______________________________________                                    

These characteristics were observed after 14 days of incubation at 25°C. The color descriptions were based on the Methuen Handbook of Colour(Kornerup, A. and Wanscher, J. H.: "Methuen Handbook of Colour," 3rded., Methuen, London (1983)).

A culture of Coelomycetes strain F-11899 thus named has been depositedwith the Fermentation Research Institute Agency of Industrial Scienceand Technology (1-3, Higashi 1 chome, Tsukuba-shi, IBARAKI 305 JAPAN) onOct. 26, 1989 under the number of FERM BP-2635.

After further studies of the classification of the strain F-11899, thestrain F-11899 more closely resembled Coleophoma empetri (Rostrup)Petrak 1929 (von Arx, J. A.: "The Genera of Fungi--Sporulating in PureCulture," 3rd ed., J. Cramer, ed., Vaduz, 1974; Sutton, B. C.: "TheCoelomycetes--Fungi Imperfecti with Pycnidia, Acervuli and Stromata,"Commonwealth Mycological Institute, Kew, 1980; Hawksworth, D. L.,Sutton, B. C., and Ainsworth, G. C.: "Dictionary of the Fungi," 7th ed.,Commonwealth Mycological Institute, Kew, 1983) belonging to the orderCoelomycetes, but differed in some pycnidial characteristics: globose orflattened at the base, immersed, and not papillate.

Considering these characteristics, we classified this strain in moredetail and renamed it as "Coleophoma sp. F-11899".

In this connection, appropriate steps were taken to amend the name,"Coelomycetes strain F-11899" to Coleophoma sp. F-11899 with theFermentation Research Institute Agency of Industrial Science andTechnology on Sep. 21, 1990.

(ii) Production of the compound [Ia] or a salt thereof

The compound [Ia] of the present invention or a salt thereof (SEQ IDNO: 1) is produced when the strain belonging to the genus Coleophomacapable of producing the compound [Ia] or a salt thereof is grown in anutrient medium containing sources of assimilable carbon and nitrogenunder aerobic conditions (e.g., shaking culture, submerged culture,etc.).

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, sucrose, starch, fructose and/or glycerin, and thelike.

The preferred sources of nitrogen are yeast extract, peptone, glutenmeal, cotton seed flour, soybean meal, corn steep liquor, dried yeast,wheat germ, etc., as well as inorganic and organic nitrogen compoundssuch as ammonium salts (e.g., ammonium nitrate, ammonium sulfate,ammonium phosphate, etc.), urea and/or amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not to be used in their pure form because less purematerials, which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such assodium or calcium carbonate, sodium or potassium phosphate, sodium orpotassium chloride, sodium or potassium iodide, magnesium salts, coppersalts, zinc salts and/or cobalt salts, and the like.

If necessary, a defoaming agent, such as liquid paraffin, fatty oil,plant oil, mineral oil or silicone, or the like may be added, especiallywhen foaming of the culture medium presents a serious problem.

As in the case of the preferred methods used for the production of otherbiologically active substances in massive amounts, submerged aerobiccultural conditions are preferred for the production of the compound[Ia] or a salt thereof in massive amounts.

For the production of the compound [Ia] or a salt thereof in smallamounts, a shaking or surface culture in a flask or bottle is employed.

Further, when the growth is carried out in large tanks, it is preferableto use the vegetative form of the organism for inoculation in theproduction tanks in order to avoid growth lag in the process ofproduction of the compound [Ia] or a salt thereof. Accordingly, it isdesirable first to produce a vegetative inoculum of the organism byinoculating a relatively small quantity of culture medium with spores ormycelia of the organism and culturing said inoculated medium, and thento transfer the cultured vegetative inoculum to large tanks. The medium,in which the vegetative inoculum is produced, is substantially the sameas the medium utilized for the production of the compound [Ia] or a saltthereof, or may be different from the medium, as desired.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 10°C. and 40° C., preferably 20° C. to 30° C., for a period of about 50hours to 150 hours, which may be varied according to fermentationconditions and scales.

When the fermentation is completed, the compound [Ia] or a salt thereofis isolated from the culture broth by various procedures conventionallyused for recovery and purification of biologically active substances.For instance, solvent extraction with an appropriate solvent or amixture of solvents, chromatography or recrystallization from anappropriate solvent or a mixture of solvents, or the like, may be used.

According to the present invention, in general, the compound [Ia] or asalt thereof is found both in the cultured mycelia and cultured broth.Accordingly, the compound [Ia] or a salt thereof is removed from thewhole broth by means of extraction using an appropriate organic solventsuch as acetone or ethyl acetate, or a mixture of these solvents, or thelike.

The extract is treated in a conventional manner to provide the compound[Ia] or a salt thereof. For example, the extract is concentrated byevaporation or distillation, and the resulting residue containing activematerial (i.e., the compound [Ia] or a salt thereof) is purified byconventional purification procedures; for example, chromatography, orrecrystallization from an appropriate solvent or a mixture of solvents,or both.

When the object compound is isolated as a salt of the compound [Ia], itcan be converted to the free compound [Ia] or another salt of thecompound [Ia] according to a conventional manner.

Process 2

The compound [Ib] (SEQ ID NO: 1) or a salt thereof can be prepared bysubjecting the compound [Ia] or a salt thereof to an eliminationreaction of the sulfo group.

Suitable salts of the compound [Ib] include the acid addition salts asexemplified for the compound [I].

This elimination reaction is carried out in accordance with conventionalmethods in this field of the art, such as reaction with an enzyme, orthe like.

The reaction with an enzyme can be carried out by reacting the compound[Ia] or a salt thereof with an enzyme suitable for the eliminationreaction of the sulfo group.

A suitable example of said enzyme includes a sulfatase, such assulfatase Type IV, produced by Aeobacter aerogenes, or the like.

This elimination reaction is usually carried out in a solvent such asphosphate buffer, Tris-HCl buffer, or any other solvent which does notadversely influence the reaction.

The reaction temperature is not critical and the reaction can be carriedout at room temperature or with warming.

Process 3

The object compound [Id] (SEQ ID NO: 1) or a salt thereof can beprepared by subjecting the compound [Ic] or a salt thereof to anelimination reaction of the appropriate N-acyl group.

This reaction is carried out in accordance with conventional methods,such as hydrolysis, reduction, reaction with an enzyme, or the like.

The hydrolysis is preferably carried out in the presence of either abase or an acid, including a Lewis acid. Suitable bases includeinorganic bases such as an alkali metal [e.g., sodium, potassium, etc.],an alkaline earth metal [e.g., magnesium, calcium, etc.], thehydroxides, carbonates or bicarbonates thereof; and organic bases, suchas trialkylamines [e.g., trimethylamine, triethylamine, etc.], picoline,1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane,1,8-diazabicyclo[5.4.0]undec-7-ene, and the like.

Suitable acids include organic acids [e.g., formic acid, acetic acid,propionic acid, trichloroacetic acid, trifluoroacetic acid, etc.] andinorganic acids [e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, etc.]. The elimination using a Lewis acid such as a trihaloaceticacid [e.g., trichloroacetic acid, trifluoroacetic acid, etc.] or thelike is preferably carried out in the presence of cation trapping agents[e.g., anisole, phenol, etc.].

The reaction is usually carried out in a solvent such as water, a loweralcohol [e.g., methanol, ethanol, etc.], methylene chloride,tetrahydrofuran, a mixture thereof or any other solvent which does notadversely influence the reaction. A liquid base or acid can be also usedas the solvent. The reaction temperature is not critical, and can becarried out either under cooling, at ambient temperatures or withwarming; for example at a temperature of from 0° C. to 100° C.

The reduction methods applicable for the elimination reaction includeboth chemical reduction and catalytic reduction.

Suitable reducing agents to be used in chemical reduction are acombination of a metal [e.g., tin, zinc, iron, etc.] or a metalliccompound [e.g., chromium chloride, chromium acetate, etc.] and anorganic or inorganic acid [e.g., formic acid, acetic acid, propionicacid, trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid,hydrobromic acid, etc.].

Suitable catalysts to be used in catalytic reduction are conventionalones such as platinum catalysts [e.g., platinum plate, spongy platinum,platinum black, colloidal platinum, platinum oxide, platinum wire,etc.], palladium catalysts [e.g., spongy palladium, palladium black,palladium oxide, palladium on carbon, colloidal palladium, palladium onbarium sulfate, palladium on barium carbonate, etc.], nickel catalysts[e.g., reduced nickel, nickel oxide, Raney nickel, etc.], cobaltcatalysts [e.g., reduced cobalt, Raney cobalt, etc.], iron catalysts[e.g., reduced iron, Raney iron, etc.], copper catalysts [e.g., reducedcopper, Raney copper, Ullman copper, etc.], and the like.

The reduction is usually carried out in a conventional solvent whichdoes not adversely influence the reaction, such as water, methanol,ethanol, propanol, N,N-dimethylformamide, or mixtures thereof.Additionally, the above-mentioned acids which can to be used in chemicalreduction which are also liquid at the reduction temperature can also beused as a solvent. Further, a suitable solvent to be used in catalyticreduction may be selected from the above-mentioned solvents and otherconventional solvents such as diethyl ether, dioxane, tetrahydrofuran,etc., or mixtures thereof.

The reaction temperature of the reduction is not critical, and thereaction is usually carried out under cooling, at ambient temperatures,or with warming; preferably, at a temperature of from 0° C. to 100° C.

The reaction with an enzyme can be carried out by reacting the compound[Ic] or a salt thereof with an enzyme suitable for the eliminationreaction of the N-acyl group.

Suitable examples of said enzyme include appropriate enzymes produced bycertain microorganisms of the Actinoplanaceae; for example, Actinoplanesutahensis IFO-13244, Actinoplanes utahensis ATCC 12301, and Actinopanesmissourienses NRRL 12053; and the like.

The enzymatic elimination reaction is usually carried out in a solventsuch as phosphate buffer, Tris-HCl buffer or any other solvent whichdoes not adversely influence the reaction.

The reaction temperature is not critical, and the reaction can becarried out at room temperature or under warming.

Process 4

The object compound [Ie] (SEQ ID NO: 1) or a salt thereof can beprepared by subjecting the compound [Id] or a salt thereof to anacylation reaction.

The acylation reaction of this process can be carried ut by reacting thecompound [Id] or a salt thereof with the aforesaid "acylating agent";for example, the compound [V], a salt thereof, or a correspondingreactive derivative at the carboxy group.

Suitable reactive derivatives at the carboxy group of the compound [V]include an acid halide or an acid anhydride (as described above), anactivated amide, an activated ester, and the like. Suitable examples ofthe reactive derivatives include an acid chloride; an acid azide; amixed acid anhydride with an acid such as a substituted phosphoric acid[e.g., dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoricacid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.],dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuricacid, sulfonic acid [e.g., methanesulfonic acid, etc.], aliphaticcarboxylic acid [e.g., acetic acid, propionic acid, butyric acid,isobutyric acid, pivalic acid, pentanoic acid, isopentanoic acid,2-ethylbutyric acid, trichloroacetic acid, etc.], or aromatic carboxylicacid [e.g., benzoic acid, etc.], a symmetrical acid anhydride; anactivated amide with imidazole, 4-substituted imidazole,dimethylpyrazole, triazole, tetrazole or 1-hydroxy-1H-benzotriazole; oran activated ester [e.g., cyanomethyl ester, methoxymethyl ester,dimethyliminomethyl [(CH₃)₂ N⁺ =CH-] ester, vinyl ester, propargylester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenylester, pentachlorophenyl ester, mesylphenyl ester, phenylazophenylester, phenyl thioester, p-nitrophenyl thioester, p-cresyl thioester,carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester,8-quinolyl thioester, etc.], or an ester with an N-hydroxy compound[e.g., N,N-dimethylhydroxylamine, 1-hydroxy-2-(1H)-pyridone,N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-1H-benzotriazole,etc.], and the like.

Suitable salts of the compound [V] and its reactive derivative are asdescribed for the compound [I].

The reaction is usually carried out in a conventional solvent such aswater, lower alcohol [e.g., methanol, ethanol, etc.], acetone, dioxane,acetonitrile, chloroform, methylene chloride, ethylene chloride,tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or anyother organic solvent which does not adversely influence the reaction.These conventional solvents may also be used in a mixture with water.

In this reaction, when the compound [V] is used in a free acid form orits salt form, the reaction is preferably carried out in the presence ofa conventional condensing agent such as N,N'-dicyclohexylcarbodiimide;N-cyclohexyl-N'-morpholinoethylcarbodiimide;N-cyclohexyl-N'-(4-diethylaminocyclohexyl)carbodiimide;N,N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide;N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide,N,N'-carbonylbis-(2-methylimidazole);pentamethyleneketene-N-cyclohexylimine;diphenylketene-N-cyclohexylimine; ethoxyacetylene;1-alkoxy-1chloroethylene; trialkyl phosphite; ethyl polyphosphate;isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride);phosphorus trichloride; thionyl chloride; oxalyl chloride; lower alkylhaloformate [e.g., ethyl chloroformate, isopropyl chloroformate, etc.];triphenylphosphine; 2-ethyl-7-hydroxybenzisoxazolium salt;2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide intramolecular salt;1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole; a Vilsmeierreagent prepared by the reaction of N,N-dimethylformamide with thionylchloride, phosgene, trichloromethyl chloroformate, phosphorusoxychloride, methanesulfonyl chloride, etc.; or the like.

The reaction may also be carried out in the presence of an inorganic ororganic base such as an alkali metal carbonate, alkali metalbicarbonate, tri(lower)alkylamine, pyridine, di(lower)alkylaminopyridine(e.g., 4-dimethylaminopyridine, etc.), N-(lower)alkylmorpholine,N,N-di(lower)alkylbenzylamine, or the like.

The reaction temperature is not critical, and the reaction can becarried out under cooling, at ambient temperatures, or with warming,preferably at a temperature of from 0° C. to 100° C.

Process 5

The object compound [Ig] (SEQ ID NO: 1) or a salt thereof can beprepared by subjecting a compound [If] or a salt thereof to anelimination reaction of the appropriate amino protective group.

Suitable salts of the compounds [If] and [Ig] can be chosen from amongthose exemplified for the compound [I]. This elimination reaction can becarried out in accordance with conventional methods, as explained abovefor Process 3.

Process 6

The object compound [Ii] (SEQ ID NO: 1) or a salt thereof can beprepared by reacting a compound [Ih] or a salt thereof with a compound[II] or a salt thereof.

Suitable salts of the compound [Ii] can be selected from the ones asexemplified for the compound [I].

Suitable salts of the compound [II] can be selected from acid additionsalts as exemplified for the compound [I].

The present reaction may be carried out in a solvent such as water,phosphate buffer, acetone, chloroform, acetonitrile, nitrobenzene,methylene chloride, ethylene chloride, formamide, N,N-dimethylformamide,methanol, ethanol, diethyl ether, tetrahydrofuran, dimethyl sulfoxide,or any other organic solvent which does not adversely affect thereaction. Preferably, the solvent has a strong polarity. Among thesolvents, hydrophilic solvents may be used in a mixture with water. Whenthe compound [II] is liquid, it can also be used as a solvent.

The reaction is preferably conducted in the presence of a base. Forexample, inorganic bases such as alkali metal hydroxides, alkali metalcarbonates, alkali metal bicarbonates, and organic bases such as atri(lower)alkylamine, and the like, are particularly suitable.

The reaction temperature is not critical, and the reaction can becarried out under cooling, at room temperature, under warming or underheating, preferably at a temperature of from 0° C. to 150° C.

The present reaction is preferably carried out in the presence of alkalimetal halide [e.g., sodium iodide, potassium iodide, etc.], alkali metalthiocyanate [e.g., sodium thiocyanate, potassium thiocyanate, etc.], orthe like.

Process 7

The object compound [Ij] (SEQ ID NO: 1) or a salt thereof can beprepared by subjecting a compound [III] or a salt thereof to anacylation reaction.

Suitable salts of the compounds [Ii] and [III] can be selected fromthose exemplified for the compound [I].

A suitable "acylating agent" in this process may be an acid compoundcorresponding to the functional group to be introduced; for example,phosphoric acid and its derivatives (e.g., phophoryl chloride,diphenylphosphorochloridate, etc.), sulfuric acid and its derivatives[e.g., sulfur trioxide-pyridine, sulfur trioxide-tri(lower)alkylamine(e.g., trimethylamine, triethylamine, etc.), chlorosulfonic acid, etc.],or the like.

This reaction can be carried out in a conventional manner.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention, are not intended to be limiting thereof.

The following Preparations and Examples are given for the purpose ofillustrating the present invention in more detail. By procedure(s) ormethod(s) similar to that of another Preparation or Example, theprocedural steps are the same, but the starting material and/orco-reactant is changed to correspond to and provide the given product.

Preparation 1

To methanol (50 ml) was added thionyl chloride (8.73 ml) at -5° C. andthe mixture was stirred for 10 minutes. D-2-(p-Hydroxyphenyl)glycine (5g) was then added thereto under ice-cooling. The mixture was stirred for12 hours at room temperature, then the volatile components wereevaporated under reduced pressure to give D-2-(p-hydroxyphenyl)glycinemethyl ester hydrochloride (6.3 g).

IR (Nujol): 3380, 1720, 1580, 1250 cm⁻¹

NMR (DMSO-d₆, δ): 3.70 (3H, s), 5.11 (1H, s), 6.83 (2H, d, J=8.6 Hz),7.28 (2H, d, J=8.6 Hz), 8.91 (2H, s), 9.93 (1H, s)

Preparation 2

To a solution of D-2-(p-hydroxyphenyl)glycine methyl ester hydrochloride(6.3 g) and triethylamine (8.71 ml ) in tetrahydrofuran (100 ml ) wasadded di-t-butyl dicarbonate (6.82 g). The mixture was stirred for 2hours at room temperature, then the reaction mixture was added todiethyl ether (1 l), and an insoluble material was filtered off. Thefiltrate was evaporated under reduced pressure to giveN-(t-butoxycarbonyl)-D-2-(p-hydroxyphenyl)glycine methyl ester (6.83 g).

IR (Nujol): 3420, 3350, 1720, 1660 cm⁻¹

NMR (DMSO-d₆, δ): 1.38 (9H, s), 3.59 (3H, s), 5.05 (1H, d, J=7.9 Hz),6.70 (2H, d, J=8.5 Hz), 7.16 (2H, d, J=8.5 Hz), 7.60 (1H, d, J=7.9 Hz),9.48 (1H, s)

Preparation 3

To a suspension of N-(t-butoxycarbonyl)-D-2-(p-hydroxyphenyl)glycinemethyl ester (6.8 g) and potassium bicarbonate (1.84 g) inN,N-dimethylformamide (34 ml) was added octyl bromide (4.176 ml). Themixture was stirred for 6 hours at 60° C. The reaction mixture was addedto a mixture of water and ethyl acetate. The organic layer wasseparated, and dried over magnesium sulfate. The magnesium sulfate wasfiltered off, and the filtrate was evaporated under reduced pressure togive N-(t-butoxycarbonyl)-D-2-(p-octyloxyphenyl)glycine methyl ester(6.96 g).

IR (Nujol): 1710, 1490, 1240, 1160 cm^(-l)

NMR (DMSO-d₆, δ): 0.859 (3H, t, J=6.2 Hz), 1.17-1.33 (10H, m), 1.38 (9H,s), 1.60-1.80 (2H, m), 3.59 (3H, s), 3.93 (2H, t, J=6.3 Hz), 5.11 (1H,d, J=7.9 Hz), 6.87 (2H, d, J=8.7 Hz), 7.27 (2H, d, J=8.7 Hz), 7.68 (1H,d, J=7.9 Hz)

Preparation 4

To a 4N aqueous solution of sodium hydroxide (8.77 ml) was addedN-(t-butoxycarbonyl)-D-2-(p-octyloxyphenyl)glycine methyl ester (6.9 g).The mixture was stirred for 1.5 hours at room temperature. The reactionmixture was added to a mixture of water and ethyl acetate, and 1Nhydrochloric acid was added thereto to adjust the mixture to pH 3. Theorganic layer was separated and dried over magnesium sulfate. Themagnesium sulfate was filtered off, and the filtrate was evaporatedunder reduced pressure to giveN-(t-butoxycarbonyl)-D-2-(p-octyloxyphenyl)glycine (3.9 g).

NMR (DMSO-d₆, δ): 0.860 (3H, t, J=6.8 Hz), 1.17-1.33 (10H, m), 1.38 (9H,s), 1.60-1.80 (2H, m), 3.93 (2H, t, J=6.4 Hz), 5.10 (1H, d, J=8.2 Hz),6.87 (2H, d, J=8.7 Hz), 7.28 (2H, d, J=8.7 Hz), 7.46 (1H, d, J=8.2 Hz)

Preparation 5

To a solution of N-(t-butoxycarbonyl)-D-2-(p-octylaxyphenyl)glycine (1g) in acetonitrile (10 ml) and pyridine (0.213 ml) in acetonitrile (10ml) was added N,N'-disuccinimidyl carbonate (0.675 g). The mixture wasstirred for 12 hours at room temperature, then was added to a mixture ofwater and ethyl acetate. The organic layer was separated and dried overmagnesium sulfate. The magnesium sulfate was filtered off, and thefiltrate was evaporated under reduced pressure to giveN-(t-butoxycarbonyl)-D-2-(p-octyloxyphenyl)glycine succinimido ester(0.92 g).

IR (Nujol): 3350, 1810, 1730, 1680 cm⁻¹

NMR (DMSO-d₆, δ): 0.862 (3H, t, J=6.7 Hz), 1.17-1.33 (10H, m), 1.40 (9H,s), 1.60-1.80 (2H, m), 2.77 (4H, s), 3.97 (2H, t, J=6.5 Hz), 5.54 (1H,d, J=8.1 Hz), 6.91 (2H, d, J=8.7 Hz), 7.39 (2H, d, J=8.7 Hz ), 8.05 (1H, d, J=8.1 Hz )

Preparations 6 through 9 were conducted in the manner of Preparations 2through 5, employing L-tyrosine methyl ester as the starting material.

Preparation 6

N-(t-Butoxycarbonyl)-L-tyrosine methyl ester was prepared by theprocedure of Preparation 2.

IR (Nujol): 3430, 3360, 1730, 1670, 1170 cm⁻¹

NMR (DMSO-d₆, δ): 1.33 (9H, s) , 2.90 (2H, m) , 3.59 (3H, s), 4.05 (1H,m), 6.65 (2H, d, J=8.4 Hz), 7.00 (2H, d, J=8.4 Hz), 7.21 (1H, d, J=8.0Hz), 9.22 (1H, s)

Preparation 7

O⁴ -Octyl-N-(t-butoxycarbonyl)-L-tyrosine methyl ester was preparedaccording to the procedure of Preparation 3.

IR (Nujol): 3350, 1735, 1685, 1250, 1170

NMR (DMSO-d₆, δ): 0.859 (3H, t, J=6.7 Hz), 1.20-1.30 (10H, m), 1.68 (2H,quintet, J=7.3 Hz), 2.82 (2H, m), 3.60 (3H, s), 3.91 (2H, t, J=7.3 Hz),4.08 (1H, m), 6.81 (2H, d, J=8.6 Hz), 7.12 (2H, d, J=8.6 Hz), 7.25 (1H,d, J=8.0 Hz)

Preparation 8

O⁴ -Octyl-N-(t-butoxycarbonyl)-L-tyrosine was prepared according to theprocedure of Preparation 4.

IR (Nujol): 3400-2900 (br), 1700, 1240, 1160 cm⁻¹

NMR (DMSO-d₆, δ): 0.859 (3H, t, J=6.8 Hz), 1.20-1.30 (10H, m), 1.32 (9H,s), 1.68 (2H, quintet, J=7.0 Hz), 2.67-2.95 (1H, m), 3.90 (2H, t, J=7.0Hz), 4.01 (1H, m), 6.81 (2H, d, J=8.6 Hz), 7.02 (1H, d, J=8.3 Hz) , 7.13(2H, d, J=8.6 Hz)

Preparation 9

O⁴ -Octyl-N- (t-butoxycarbonyl) -L-tyrosine succinimido ester wasprepared according to the procedure of Preparation 5.

IR (Nujol): 3350, 1780, 1720, 1690 cm⁻¹

NMR (DMSO-d₆, δ): 0.860 (3H, t, J=6.7 Hz), 1.20-1.30 (10H, m), 1.32 (9H,s), 1.68 (2H, quintet, J=7.0 Hz), 2.82 (4H, s), 2.80-3.20 (1H, m), 3.92(2H, t, J=7.0 Hz), 4.44 (1H, m), 6.81 (2H, d, J=8.5 Hz), 7.22 (2H, d,J=8.5 Hz), 7.60 (1H, d, J=8.3 Hz)

Preparation 10

(1) A seed medium (160 ml) consisting of 4% sucrose, 2% cotton seedflour, 1% dried yeast, 1% peptone, 0.2% KH₂ PO₄, 0.2% CaCO₃ and 0.1%TWEEN 80 (made by NAKARAI CHEMICALS LTD.) was poured into each of two500 ml Erlenmeyer flasks and sterilized at 121° C. for 30 minutes. Aloopful of slant culture of Coleophoma sp. F-11899 was inoculated toeach of the media and cultured with shaking at 25° C. for 4 days.

A production medium (20 liters) consisting of 3% PINE DEX #3 (made byMatsutani Chemical Ltd.), 1% glucose, 1% wheat germ, 0.5% cotton seedflour, 2% KH₂ PO₄, 1.5% Na₂ HPO₄.12H₂ O, 0.001% ZnSO₄.7H₂ O and 0.05%ADEKANOL (defoaming agent, made by Asahi Denka Co., Ltd.) was pouredinto a 30 liter jar fermentor and sterilized at 121° C. for 30 minutes.

The resultant seed culture broth (320 ml) was inoculated to theproduction medium and cultured at 25° C. for 4 days, agitated at 200 rpmand aerated at 20 liters per minute. To the cultured broth thus obtained(20 liters) was added an equal volume of acetone. After occasionallystirring at room temperature for a while, the broth was filtered. Thefiltrate was concentrated in vacuo to remove acetone. The aqueousfiltrate (10 liters) was washed with two equal volumes of ethyl acetate,and extracted with n-butanol (10 liters) twice. The combined n-butanollayers were concentrated in vacuo and the residue was applied on acolumn (300 ml) of SILICA GEL 60 (made by E. Merck) and eluted with astepwise organic solvent mixture consisting of dichloromethane-methanol.The fractions having anti-Candida activity were eluted in the range ofthe solvent mixture (3:1 through 1:1). The active fractions werecombined and concentrated in vacuo to dryness. The residue was dissolvedin 50% aqueous methanol (15 ml) and applied on a column (250 ml) of ODSYMC GEL (made by Yamamura Chemical Lab.). The column was washed with 50%aqueous methanol and eluted with 80% aqueous methanol. The eluate wasconcentrated and was further purified on a centrifugal partitionchromatography (CPC) using a solvent system n-butanol:methanol:water(4:1:5) of upper stationary phase and lower mobile phase in a descendingmode. The pooled fractions containing the object compound (majorcomponent) were concentrated in vacuo and applied on a column (35 ml) ofSILICA GEL 60. The column was developed with n-butanol:acetic acid:water(6:1:1). The active fractions were combined and concentrated in vacuo todryness and dissolved in a small volume of 50% aqueous methanol. Thesolution was passed through a column (3.5 ml) of ODS YME GEL. The columnwas washed with 50% aqueous methanol and eluted with methanol. Theeluate was concentrated to dryness, dissolved in a small volume of waterand adjusted to pH 7.0 with 0.01N NaOH. The solution was freeze-dried togive a white powder in its sodium salt form (hereinafter referred to asFR901379 substance) (11 mg).

The fractions containing two minor components after CPC wereconcentrated in vacuo and purified on a preparative high performanceliquid chromatography (HPLC) column of LICHROSORB RP-18 (Trademark, madeby Merck, 250×25 mm) using a mobile phase composed of 45% aqueous CH₃CN-0.5% NH₄ H₂ PO₄ at a flow rate of 9.9 ml/minute. The fractioncontaining one of the two components was diluted with an equal volume ofwater and passed through a column (1 ml) of ODS YMC Gel. The column waswashed with 40% aqueous MeOH and eluted with MeOH. The eluate wasconcentrated in vacuo to dryness, then dissolved in a small volume ofwater and freeze-dried to give a white powder in its ammonium salt form(2.2 mg) (hereinafter referred to as FR901381 substance).

In a similar manner, the other minor component in its ammonium salt formwas obtained as a white powder (1.2 mg) (hereinafter referred to asFR901382 substance).

The FR901379 substance as obtained has the following physico-chemicalproperties:

Appearance:

white powder

Nature:

neutral substance

Melting point:

215°-221° C. (dec.)

Specific rotation:

[a]_(D) ²³ -20.3 (C: 0.5, H₂ O)

Molecular formula:

C₅₁ H₈₁ N₈ O₂₁ SNa

Elemental Analysis:

Calcd: for C₅₁ H₈₁ N₈ SO₂₁ Na C 51.17, H 6.77, N 9.36, S 2.68 (%)

Found: C 49.61, H 7.58, N 7.65, S 2.14 (%)

Molecular weight:

HRFAB-MS 1219.5078 (Calcd for C₅₁ H₈₂ N₈ SO₂₁ +2Na - H: 1219.5032)

Solubility:

soluble: methanol, water

slightly soluble: ethyl acetate, acetone

insoluble: chloroform, n-hexane

Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction, ferricchloride reaction, Ninhydrin reaction

negative: Dragendorff reaction, Ehrlich reaction

Thin layer chromatography (TLC):

    ______________________________________                                        Stationary phase                                                                            Developing Solvent                                                                           Rf value                                         ______________________________________                                        silica gel*   N-butanol:acetic acid:                                                                       0.36                                                           water (3:1:1)                                                                 ethyl acetate:isopropyl                                                                      0.31                                                           alcohol:water (5:3:1)                                           ______________________________________                                         *SILICA GEL 60 (made by E. Merck)                                        

Ultraviolet absorption spectrum:

k_(max) ^(methanol) (E_(1cm) ^(1%) ): 207(169), 276(13.5), 225(sh),283(sh) nm

k_(max) ^(methanol+0).01N-NaOH (E_(1cm) ^(1%)): 209(232), 244(59.5),284(13.5), 294(sh) nm

Infrared absorption spectrum:

t_(max) ^(KBr) : 3350, 2920, 2840, 1660, 1625, 1530, 1510, 1435, 1270,1240, 1070, 1045, 800, 755, 710 cm⁻¹

¹ H Nuclear magnetic resonance spectrum:

(CD₃ OD, 400 MHz)

δ: 7.30 (1H, d, J=2 Hz), 7.03 (1H, dd, J=8 and 2 Hz), 6.85 (1H, d, J=8Hz), 5.23 (1H, d, J=3 Hz), 5.06 (1H, d, J=4 Hz), 4.93 (1H, d, J=3 Hz),4.59-4.51 (3H, m), 4.47-4.35 (5H, m), 4.29 (1H, dd, J=6 and 2 Hz ), 4.17( 1H, m ), 4.07 ( 1H, m ), 3.95-3.89 (2H, m), 3.76 (1H, broad d, J=11Hz), 3.36 (1H, m), 2.75 (1H, dd, J=16 and 4 Hz), 2.50 (1H, m), 2.47 (1H,dd, J=16 and 9 Hz), 2.38 (1H, m), 2.21 (2H, m), 2.03-1.93 (3H, m), 1.57(2H, m), 1.45-1.20 (24H, m), 1.19 (3H, d, J=6 Hz), 1.08 (3H, d, J=6 Hz),0.90 (3H, t, J=7 Hz)

From the analysis of the above physical and chemical properties, and theresult of the further investigation and identification of the chemicalstructure, the chemical structure of the FR901379 substance has beenidentified and assigned as follows (SEQ ID NO: 1): ##STR8##

The FR901381 substance as obtained has the following physico-chemicalproperties:

Appearance:

white powder

Nature:

neutral substance

Melting point:

218°-223° C. (dec.)

Specific rotation:

[a]_(D) ²³ -10.5° (C: 0.5, MeOH)

Molecular formula:

C₅₁ H₈₁ N₈ O₂₀ S·NH₄

Molecular weight:

HRFAB-MS 1203.5100

(Calcd for C₅₁ H₈₂ N₈ O₂₀ S+2Na-H: 1203.5083)

Solubility:

soluble: methanol, ethanol

slightly soluble: water, acetone

insoluble: chloroform, n-hexane

Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction

negative: Dragendorff reaction, Ehrlich reaction

Thin layer chromatography (TLC):

    ______________________________________                                        Stationary phase                                                                            Developing Solvent                                                                           Rf value                                         ______________________________________                                        silica gel*   N-butanol:acetic acid:                                                                       0.34                                                           water (3:1:1)                                                                 ethyl acetate:isopropyl                                                                      0.67                                                           alcohol:water (5:3:1)                                           ______________________________________                                         *SILICA GEL 60 (made by E. Merck)                                        

Ultraviolet absorption spectrum:

k_(max) ^(methanol) (E_(1cm) ^(1%)): 206(196), 278(4), 243(sh), 284(sh)nm

k_(max) ^(methanol+0).01 N-NaOH E_(1cm) ^(1%)): 208(252), 290(5),241(sh) nm

Infrared absorption spectrum:

t_(max) ^(KBr) : 3300, 2900, 2840, 1680, 1660, 1640, 1620, 1510, 1460,1430, 1330, 1240, 1040, 960 cm⁻¹

¹ H Nuclear magnetic resonance spectrum:

(CD₃ OD, 400 MHz)

δ: 7.18 (1H, d, J=2 Hz), 6.90 (1H, dd, J=2 and 8.5 Hz), 6.81 (1H, d,J=8.5 Hz), 5.29 (1H, d, J=3 Hz), 5.08 (1H, d, J=3.5 Hz), 4.98 (1H, d,J=3 Hz), 4.63 (1H, dd, J=7 and 11 Hz), 4.58-4.51 (3H, m), 4.46-4.38 (3H,m), 4.37 (1H, d, J=2 Hz), 4.16 (1H, dd, J=2 and 5 Hz), 4.07 (1H, dd,J=7.5 and 9.5 Hz), 4.02-3.94 (2H, m), 3.78 (1H, br d, J=11 Hz), 3.38(1H, t, J=9.5 Hz), 2.69 (1H, dd, J=4.5 and 15 Hz), 2.63-2.50 (3H, m),2.46 (1H, m), 2.43 (1H, dd, J=9 and 15 Hz), 2.21 (2H, t, J=7.5 Hz),2.07-1.95 (3H, m), 1.58 (2H, m), 1.29 (24H, m), 1.16 (3H, d, J=6.8 Hz),1.07 (3H, d, J=7 Hz), 0.89 (3H, t, J=6.5 Hz)

¹³ C Nuclear magnetic resonance spectrum:

(CD₃ OD, 100 MHz)

δ: 176.7 (s), 175.9 (s), 174.4 (s), 174.0 (s), 172.8 (s), 172.5 (s),172.5 (s), 169.4 (s), 149.1 (s), 141.1 (s), 131.1 (s), 128.0 (d), 125.3(d), 118.3 (d), 75.9 (d), 74.0 (d), 73.9 (d), 71.3 (d), 70.7 (d), 70.5(d), 70.2 (d), 68.2 (d), 62.4 (d), 58.6 (d), 58.4 (d), 57.2 (t), 55.5(d), 52.9 (t), 51.4 (d), 40.8 (t), 39.9 (t), 39.1 (d), 39.0 (t), 36.7(t), 35.0 (t), 33.1 (t), 30.8 (t×5), 30.7 (t), 30.7 (t), 30.5 (t), 30.4(t), 30.3 (t), 27.0 (t), 23.7 (t), 19.5 (q), 14.4 (g), 11.1 (q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation for identification of the chemicalstructure, the chemical structure of the FR901381 substance has beenidentified and assigned as follows (SEQ ID NO: 1): ##STR9##

The FR901382 substance as obtained has the following physico-chemicalproperties:

Appearance:

white powder

Nature:

neutral substance

Melting point:

208°-217° C. (dec.)

Specific rotation:

[a]_(D) ²³ -9.4° (C: 0.5, MeOH).

Molecular formula:

C₅₁ H₈₁ N₈ O₁₉ S·NH₄

Molecular weight:

HRFAB-MS 1187.5139

(Calcd. for C₅₁ H₈₂ N₈ O₁₉ S+2Na-H 1187.5134)

Solubility:

soluble: methanol, ethanol

slightly soluble: water, acetone

insoluble: chloroform, n-hexane

Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction

negative: Dragendorff reaction, Ehrlich reaction

Thin layer chromatography (TLC):

    ______________________________________                                        Stationary phase                                                                            Developing Solvent                                                                           Rf value                                         ______________________________________                                        silica gel*   N-butanol:acetic acid:                                                                       0.43                                                           water (3:1:1)                                                                 ethyl acetate:isopropyl                                                                      0.9                                                            alcohol:water (5:3:1)                                           ______________________________________                                         *SILICA GEL 60 (made by E. Merck)                                        

Ultraviolet absorption spectrum:

k_(max) ^(methanol) (E_(1cm) ^(1%)): 205(180), 276(13) 224(sh), 283 (sh)nm

k_(max) ^(methanol+0).01N-NaOH (E_(1cm) ^(1%)): 208(262), 281(12),241(sh), 295(sh) nm

Infrared absorption spectrum:

t_(max) ^(KBr) : 3350, 2900, 2840, 1680, 1660, 1640, 1620, 1510, 1430,1330, 1245, 1080, 1040, 960 cm⁻

¹ H Nuclear magnetic resonance spectrum:

(CH₃ OD, 400 MHz)

δ: 7.18 (1H, d, J=2 Hz), 6.90 (1H, dd, J=2 and 8.5 Hz), 6.80 (1H, d,J=8.5 Hz), 5.37 (1H, dd, J=3 and 11 Hz), 5.08(1H, d, J=3.5 Hz), 5.00(1H, d, J=3 Hz), 4.61 (1H, dd, J=7 and 11 Hz), 4.59 (1H, d, J=2 Hz),4.58-4.52 (2H, m), 4.46-4.35 (3H, m), 4.29 (1H, d, J=2 Hz), 4.12 (1H,dd, J=2 and 4.5 Hz), 4.07 (1H, dd, J=8 and 9.5 Hz), 4.01 (1H, dd, J=3and 11 Hz), 3.77 (1H, br d, J=11 Hz), 3.37 (1H, t, J=9.5 Hz), 2.69 (1H,dd, J=4.5 and 15.5 Hz), 2.63-2.50 (3H,m), 2.45 (1H, m), 2.43 (1H, dd,J=9 and 15.5 Hz), 2.24 (2H, m), 2.09-1.95 (3H, m), 1.76-1.66 (2H, m),1.59 (2H, m), 1.29 (24H, m), 1.15 (3H, d, J=6.5 Hz), 1.06 (3H, d, J=7Hz), 0.89 (3H, t, J=7 Hz)

¹³ C Nuclear magnetic resonance spectrum:

(CD₃ OD, 100 MHz)

δ:176.7 (s), 176.0 (s), 175.1 (s), 174.0 (s), 172.8 (s), 172.6 (s),172.5 (s), 169.1 (s), 149.1 (s), 141.1 (s), 131.1 (s), 128.1 (d), 125.3(d), 118.2 (d), 76.1 (d), 74.0 (d), 71.8 (d), 71.3 (d), 70.5 (d), 70.3(d), 68.3 (d), 62.5 (d), 58.5 (d), 58.2 (d), 57.2 (t), 55.4 (d), 52.9(t), 52.1 (d), 40.8 (t), 39.8 (t), 39.1 (d), 38.9 (t), 36.8 (t), 33.1(t), 30.9 (t), 30.8 (t×5), 30.7 (t), 30.7 (t), 30.5 (t), 30.4 (t), 30.3(t), 27.3 (t), 26.9 (t), 23.7 (t), 19.4 (q), 14.4 (q), 11.1 (q)

From the analysis of the above physical and chemical properties, and theresult of the further investigation for identification of the chemicalstructure, the chemical structure of the FR901382 substance has beenidentified and assigned as follows (SEQ ID NO: 1): ##STR10##

Preparation 10-1

To a solution of FR901379 substance (60 mg) in 50 mM Tris-HCl buffer (pH7.1, 30 ml) was added sulfatase (200 U) Type VI from Aerobacteraerogenes (SIGMA No. S-1629). After incubating at 37° C. for 30 hours,the desulfonated FR901379 substance (hereinafter referred to as FR133302substance) was extracted from the reaction mixture with a equal volumeof n-butanol, then the organic phase was separated and washed once withwater. The extract was concentrated in vacuo and applied on a column ofLICHROPREP RP-18 (40-63 μm) pre-packed size B (made by Merck),equilibrated with 47% aqueous acetonitrile containing 0.5% NH₄ H₂ PO₄,and developed with the same solution. The fraction containing FR133302substance was diluted with an equal volume of water, and directly passedthrough a column of ODS YMC GEL (made by Yamamura Chemical Lab.). Thecolumn was washed with water and eluted with methanol. The eluate wasevaporated in vacuo to remove the methanol, and freeze-dried to give awhite powder of FR133302 substance (26 mg).

The FR133302 substance has the following physico-chemical properties:

Appearance:

white powder

Nature:

neutral substance

Melting point:

218°-222° C. (dec.)

Specific rotation:

[a]_(D) ²³ -30° (C: 1.0, MeOH)

Molecular formula:

C₅₁ H₈₂ N₈ O₁₈

Molecular weight:

HRFAB-MS 1117.5659

(Calcd. for C₅₁ H₈₂ N₈ O₁₈ +Na 1117.5645)

Solubility:

soluble: methanol, ethanol

slightly soluble: water, ethyl acetate

insoluble: chloroform, n-hexane

Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction

negative: Dragendorff reaction, Molish reaction

Thin layer chromatography (TLC):

    ______________________________________                                        Stationary phase                                                                            Developing Solvent                                                                           Rf value                                         ______________________________________                                        silica gel*   n-butanol:acetic acid:                                                                       0.35                                                           water (6:1:1)                                                   ______________________________________                                         *SILICA GEL 60 (made by E. Merck)                                        

Ultraviolet absorption spectrum:

k_(max) ^(methanol) (E_(1cm) ^(1%)): 207(353), 282(25), 232(sh), nm

k_(max) ^(methanol+0).01N-NaOH (E_(1cm) ^(1%)): 208(462), 246(54.5),293(31.2) nm

Infrared absorption spectrum:

t_(max) ^(KBr) : 3350, 2925, 2855, 1660, 1630, 1530, 1445, 1285, 1250,1065 cm⁻¹

¹ H Nuclear magnetic resonance spectrum:

(CD₃ OD, 400 MHz)

δ: 6.79 (1H, d, J=2 Hz), 6.71 (1H, d, J=8 Hz), 6.61 (1H, dd, J=8 and 2Hz), 5.25 (1H, d, J=2.5 Hz), 5.06 (1H, d, J=4 Hz), 4.96 (1H, d, J=3 Hz),4.60-4.20 (9H, m), 4.15 (1H, m), 4.08 (1H, m), 3.99 (1H, m), 3.91 (1H,m), 3.77 (1H, m), 3.34 (1H, m), 2.80 (1H, dd, J=15 and 3 Hz), 2.54-2.40(3H, m), 2.20 (2H, t, J=7 Hz), 2.05-1.96 (3H, m), 1.56 (2H, m),1.35-1.20 (24H, m), 1.15 (3H, d, J=6 Hz), 1.02 (3H, d, J=7 Hz), 0.89(3H, t, J=7 Hz)

¹³ C Nuclear magnetic resonance spectrum:

(CD₃ OD, 100 MHz)

δ: 177.2 (s), 175.8 (s), 174.5 (s), 173.4 (s), 172.7 (s), 172.6 (s),172.5 (s), 169.1 (s), 146.4 (s), 146.3 (s), 133.7 (s), 120.1 (d), 116.2(d), 115.3 (d), 76.9 (d), 75.9 (d), 75.8 (d), 74.0 (d), 71.3 (d), 70.6(d), 70.6 (d), 70.1 (d), 68.2 (d), 62.5 (d), 58.4 (d), 57.1 (t), 56.4(d), 55.6 (d), 53.0 (t), 51.5 (d), 39.5 (t), 39.0 (d), 38.5 (t), 36.7(t), 34.8 (t), 33.1 (t), 30.8 (t×5), 30.7 (t), 30.6 (t), 30.5 (t), 30.4(t), 30.3 (t), 26.9 (t), 23.7 (t), 19.7 (q), 14.4 (g), 11.1 (q)

The chemical structure of the FR133302 substance is follows (SEQ ID NO:1): ##STR11##

EXAMPLE 1

The N-acyl group of FR901379 substance was eliminated by reaction withan enzyme. In the following description, this elimination process isexplained in detail.

(1) Fermentation of Actinoplanes utahensis

The enzyme which is useful for eliminating the N-acyl group of FR901379substance is produced by certain microorganisms of the Actinoplanaceae,preferably the microorganism Actinoplanes utahensis IFO-13244.

A stock culture of Actinoplanes utihensis IFO-13244 was prepared andmaintained on an agar slant. A loopful of the slant culture wasinoculated into a seed medium consisting of 1% starch, 1% sucrose, 1%glucose, 1% cotton seed flour, 0.5% peptone, 0.5% soy bean meal and 0.1%CaCO₃. The inoculated vegetative medium was incubated in a 225 ml widemouth Erlenmeyer flask at 30° C. for about 72 hours on a rotary shaker.

This incubated vegetative medium was used directly to inoculate into aproduction medium consisting of 2% sucrose, 1% peanut powder, 0.12% K₂HPO₄, 0.05% KH₂ PO₄ and 0.025% MgSO₄ ·7H₂ O. The inoculated productionmedium was allowed to ferment in a 30 liter jar fermentor at atemperature of 30° C. for about 80 hours. The fermentation medium wasstirred with conventional agitators at 250 rpm and aerated at 20 litersper minute. The vegetative mycelium was collected from the fermentedbroth by filtration and once washed with water. The washed mycelium wasdirectly used as an enzyme source to eliminate the N-acyl group ofFR901379 substance.

(2) Elimination Conditions

FR901379 substance was dissolved in 0.25M phosphate buffer (pH 6.5) at aconcentration of 0.9 mg/ml. To 36 liters of the solution, 2 kg wetweight of the washed mycelium of Actinoplanes utahensis IFO-13244 wasadded. The elimination reaction was carried out at 37° C. for 23 hours.The reduction of FR901379 substance and subsequent increase of thedeacylated FR901379 substance (hereinafter referred to as FR133303substance) were measured using a HPLC equipped with a reverse phasecolumn. From 30 g of FR901379 substance, 22.2 g of FR133303 substancewas formed in the reaction mixture.

(3) Isolation of FR133303 Substance

The reaction mixture described above was filtered with a filter aid. Themycelial cake was discarded. The filtrate thus obtained was passedthrough a column of activated carbon (2 L). The column was washed with 6L of water and eluted with 12 L of 50% aqueous acetone. The eluate wasevaporated in vacuo to remove acetone and then passed through a column(4 L) of YMC GEL ODS-AM 120-S50 (Yamamura Chemical Labs). The column waswashed with water and eluted with 2% aqueous acetonitrile containing 50mM NaH₂ PO₄. Elution was monitored by analytical HPLC, using a column ofLICHROSPHER 100 RP-18 (Cica-MERCK) and a solvent system of 3% aqueousacetonitrile containing 0.5% NH₄ H₂ PO₄ at a flow rate of 1 ml/min,detecting the FR133303 substance with a UV monitor at 210 nm. Thefractions containing the FR133303 substance were combined passed througha column of activated carbon (400 ml). The column was washed with waterand eluted with 50% aqueous acetone. The eluate was concentrated invacuo to remove acetone, and lyophilized to give 16.4 g of FR133303substance as a white powder.

FR133303 substance has following physico-chemical properties:

Appearance:

white powder

Melting point:

150°-160° C. (dec.)

Specific rotation:

[a]_(D) ²⁴ -31.17° (C: 1.0, H₂ O)

Molecular formula:

C₃₅ H₅₁ N₈ SO₂₀ Na:

Elemental Analysis:

Calcd: for C₃₅ H₅₁ N₈ SO20Na: C 43.84, H 5.36, N 11.69, S 3.34 (%)

Found: C 41.14, H 5.74, N 10.88, S 3.10 (%)

Solubility:

1soluble: water

slightly soluble: methanol

insoluble: n-hexane

Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction, Ninhydrinreaction

negative: Molish reaction

Thin layer chromatography (TLC):

    ______________________________________                                        Stationary phase                                                                            Developing Solvent                                                                           Rf value                                         ______________________________________                                        silica gel*   N-butanol:acetic acid:                                                                       0.15                                                           water (3:1:1)                                                   ______________________________________                                         *SILICA GEL 60 (made by E. Merck)                                        

Ultraviolet absorption spectrum:

k_(max) ^(H).sbsp.2^(O) (E₁ cm^(1%)): 201(340), 273(18), 224(sh),281(sh) nm

k_(max) ^(H).sbsp.2^(O+0).01 N-NaOH (E₁ cm^(1%)): 207(414), 243(122),292 (34)

Infrared absorption spectrum:

t_(max) ^(KBr) : 3350, 2920, 1660, 1625, 1515, 1440, 1270, 1080, 1045,800, 755, 715 cm⁻¹

¹ H Nuclear magnetic resonance spectrum:

(D₂ O, 400 MHz)

δ: 7.31 (1H, d, J=2 Hz), 7.12 (1H, dd, J=2 and 8 Hz), 7.06 (1H, d, J=8Hz), 5.40 (1H, d, J=3 Hz), 5.04 (1H, d, J=3.5 Hz), 4.94 (1H, d, J=6 Hz),4.73-4.55 (3H, m), 4.51-4.38 (4H, m), 4.31-4.23 (3H, m), 4.11-4.06 (2H,m), 3.94-3.89 (2H, m), 3.41 (1H, m), 2.60-2.34 (5H, m), 2.14 (1H, m),2.03 (1H, m), 1.28 (3H, d, J=6 Hz), 1.01 (3H, d, J=6.5 Hz)

¹³ C Nuclear magnetic resonance spectrum:

(D20, 100 M Hz)

δ: 178.3 (s), 175.9 (s), 174.3 (s), 174.2 (s), 174.0 (s), 171.8 (s),171.3 (s), 150.9 (s), 141.5 (s), 134.4 (s), 128.2 (d), 124.5 (d), 120.3(d), 78.1 (d), 77.0 (d), 76.9 (d), 76.6 (d), 72.9 (d), 72.8 (d), 71.2(d), 69.3 (d), 69.2 (d), 63.7 (d), 60.1 (d), 58.3 (t), 58.0 (d), 56.9(d), 55.3 (d), 54.7 (t), 41.8 (t), 39.7 (d), 39.5 (t), 33.5 (t), 21.4(q), 13.3 (q)

The chemical structure of FR133303 substance has been identified andassigned as follows (SEQ ID NO: 1): ##STR12##

EXAMPLE 2

(1) A solution of 4-hydroxybenzoic acid (19.2 g) in 10% NaOH (120 ml)was dropwise added to 480 ml of dimethyl sulfoxide over 30 minutesduring which the temeperature in reaction mixture was controlled between30° and 40° C. After adding, the solution was cooled to 17°-20° C.1-Bromooctane (28.95 g) was drowise added to the solution over 30minutes and the reaction mixture was vigorously stirred for 4 hours atroom temperature. The reaction mixture was poured into ice water (1200ml) and acidified with 40 ml of conc. hydochloric acid. After vigorouslystirring for 1 hour, the resulting solid was removed by filtration, anddissolved in 60 ml of acetonitrile. The solution was refluxed for 30minutes, then was allowed to stand overnight at room temperature toyield 4-octyloxybenzoic acid (13.8 g) as crystals (m.p. 96° C.; Anal:Calcd. for C₁₅ H₂₂ O₃ : C 71.97, H 8.86, Found: C 71.30, H 8.89).

To a solution of 4-octyloxybenzoic acid (13.8 g) in diethyl ether (552ml) were added 2,4,5-trichlorophenol (10.87 g) andN,N'-dicyclohexylcarbodiimide (11.37 g). The solution was stirred undera nitrogen atmosphere for 18 hours at room temperature. The precipitatewas removed by filtration, and the filtrate was concentrated in vacuo.The residue was dissolved in petroleum ether and was allowed to stand onice-water. The resulting crystals (15.2 g) were filtered and dissolvedin warm n-hexane (150 ml). After standing overnight at room temperature,the resulting crystals were removed by filtration. The filtrate wasconcentrated to an oil which was purified by a column chromatography(silica gel) using a mixture of ethyl acetate and n-hexane to give2,4,5-trichlorophenyl 4-octyloxybenzoate (7.58 g) (m.p. 53° C., Anal:Calcd. for C₂₁ H₂₃ O₃ Cl₃ :Cl24.75, Found: Cl 24.05).

(2) To a solution of FR133303 substance (2.04 g) inN,N-dimethylformamide (60 ml) were added 2,4,5-trichlorophenyl4-octyloxybenzoate (2.04 g) and 4-dimethylaminopyridine (0.283 g). Thesolution was stirred under a nitrogen atmosphere at room temperature for15 hours. 4-Dimethylaminopyridine (0.20 g) was added to the solution andmixture was stirred for another 24 hours. The reaction mixture waspoured into water (600 ml) and the pH was adjusted to 6.0. The mixturewas washed twice with an equal volume of ethyl acetate and concentratedto 30 ml. The concentrate was applied on a column (150 ml) ofDEAE-TOYOPEARL (Cl type, manufactured by Tosoh). The column was washedwith 50% aqueous methanol and developed with 50% aqueous methanolcontaining 1M aqueous sodium chloride. Product elution was monitored bythe same HPLC system as described in Example 1(3) except that theconcentration of acetonitrile in the solvent mixture was 40%. Thefractions containing the object compound were pooled and evaporated invacuo to remove methanol. The solution was absorbed on a column (1 L) ofYMC GEL ODS-AM 120-S50 in order to remove salt(s). The column was washedwith water and eluted with 30% aqueous acetonitrile. The eluate wasevaporated in vacuo to remove acetonitrile and lyophilized to give theobject compound (hereinafter referred to as FR131535 substance) (1.4 g)as a white powder.

FR131535 substance has following physico-chemical properties:

Appearance:

white powder

Melting point:

170°-189° C. (dec.)

Specific rotation:

[a]_(D) ²⁰ -14.4° (C: 10, H₂ O)

Molecular formula:

C₅₀ H₇₁ N₈ SO₂₂ Na

Elemental Analysis:

Calcd: for C₅₀ H₇₁ N₈ SO₂₂ NaO6H₂ O: C 46.22, H 6.44, N 8.62, S 2.46, Na1.77 (%)

Found: C 46.80, H 6.13, N 8.78, S 1.96, Na 1.81 (%)

Solubility:

soluble: methanol, water

slightly soluble: acetone

insoluble: n-hexane

Color reaction:

positive: iodine vapor reaction, cerium sulfate reaction

Thin layer chromatography (TLC):

    ______________________________________                                        Stationary phase                                                                            Developing Solvent                                                                           Rf value                                         ______________________________________                                        silica gel*   n-butanol:acetic acid:                                                                       0.21                                                           water (6:1:1)                                                   ______________________________________                                         *SILICA GEL 60 (made by E. Merck)                                        

Ultraviolet absorption spectrum:

t_(max) ^(KBr) : 3330, 2900, 2850, 1620, 1500, 1430, 1270, 1250, 1170,1110, 1080, 1040, 960, 940, 880, 840, 800, 750, 710 cm⁻¹

¹ H Nuclear magnetic resonance spectrum:

(CD₃ OD, 200 MHz)

δ: 7.78 (2H, d, J=8 Hz), 7.31 (1H, d, J=2 Hz), 7.03 (1H, dd, J=2 and 8Hz), 6.96 (2H, d, J=8 Hz), 6.87 (1H, d, J=8 Hz), 5.33 (1H, d, J=3 Hz),5.08 (1H, d, J=4 Hz), 4.99 (1H, d, J=3 Hz), 4.80-3.20 (17H, m), 2.83(1H, m), 2.65-2.30 (4H, m), 2.22-1.90 (2H, m), 1.79 (2H, m), 1.56-1.25(10H, m), 1.19 (3H, d, J=6 Hz), 1.06 (3H, d, J=6.5 Hz), 0.90 (3H, t,J=6.5 Hz)

The chemical structure of FR131535 substance has been identified andassigned as follows (SEQ ID NO: 1): ##STR13##

In the following, the structures of the compounds of Examples 3 to 11are shown. ##STR14##

    ______________________________________                                        Example                                                                              Compound                                                               No.    No.       R                                                            ______________________________________                                        3      FR138260                                                               4      FR138727                                                                                 ##STR15##                                                   5      FR138364                                                                                 ##STR16##                                                   6      FR138261  COO.sup.t Bu                                                 7      FR138363  COCH.sub.3                                                   8      FR138728  COCH.sub.2 Br                                                9      FR138538                                                                                 ##STR17##                                                   10     FR138539                                                                                 ##STR18##                                                   11     FR138365                                                                                 ##STR19##                                                   ______________________________________                                    

EXAMPLE 3

To a solution of FR133303 substance (1 g) andN-(t-butoxycarbonyl)-D-2-(p-octyloxyphenyl)glycine succinimido ester(0.596 g) in N,N-dimethylformamide (3 ml) was added4-dimethylaminopyridine (0.165 g). The mixture was stirred for 12 hoursat room temperature. The reaction mixture was added to water (30 ml),and then the pH was adjusted to 6. The aqueous solution was washed withethyl acetate, and subjected to ion exchange chromatography onDEAE-TOYOPEARL (Cl⁻)(60 ml) and eluted with 50% methanol in 1M aqueoussodium chloride. The fractions containing the object compound werecombined and evaporated under reduced pressure to remove methanol. Theaqueous solution was adjusted to pH 4.5 with 1N hydrochloric acid andsubjected to column chromatography on DIAION HP-20 (Trademark,Manufactured by Mitsubishi Chemical Industries) (130 ml) and eluted with80% aqueous methanol. The fractions containing the object compound werecombined and evaporated under reduced preessure to remove methanol. Theresidue was lyophilized to give the object acylated compound(hereinafter referred to FR138260 substance) (0.77 g).

IR (Nujol): 3300, 1660, 1500, 1240, 1045, 800, 720 cm⁻¹

NMR (CD₃ OD, δ): 0.92 (3H, t, J=6.8 Hz), 1.05 (3H, d, J=6.8 Hz),1.17-1.33 (13H, m), 1.43 (9H, s), 1.6-1.8 (2H, m), 1.9-2.1 (3H, m), 2.50(3H, m), 2.75 (1H, dd, J=16 and 4 Hz), 3.35 (1H, m), 3.7-3.8 (1H, m),3.93 (2H, t, J=6.2 Hz), 3.9-4.2 (5H, m), 4.3-4.5 (5H, m), 4.5-4.7 (3H,m), 4.97 (1H, d, J=3 Hz), 5.05 (1H, d, J=4 Hz), 5.11 (1H, s), 5.30 (1H,d, J=3 Hz), 6.85 (1H, d, J=8.3 Hz), 6.86 (2H, d, J=8.6 Hz), 7.02 (1H, d,J=8.3 Hz), 7.26 (2H, d, J=8.6 Hz), 7.31 (1H, s)

FAB-MS: e/z=1343 (M+Na)

EXAMPLE 4

FR138260 substance obtained in Example 3 (0.25 g) was added totrifluoroacetic acid (1.25 ml) and stirred for 10 minutes. The reactionmixture was added to water (30 ml) and then adjusted to pH 4.5 with asaturated aqueous solution of sodium bicarbonate. The aqueous solutionwas objected to column chromatography on DIAION HP-20 (100 ml) andeluted with 80% aqueous methanol. The fractions containing the objectcompound were combined and evaporated under reduced pressure to removemethanol. The residue was lyophilized to give the object compound(hereinafter referred to as FR138727 substance) (15 mg).

NMR (CD₃ OD, δ): 0.90 (3H, t, J=6.8 Hz), 1.05 (3H, d, J=6.8 Hz),1.17-1.33 (13H, m), 1.6-1.8 (2H, m), 1.9-2.1 (3H, m), 2.50 (1H, m), 2.75(1H, dd, J=16 and 4 Hz), 3.40 (1H, m), 3.7-3.8 (1H, m), 3.98 (2H, t,J=6.2 Hz), 3.9-4.2 (5H, m), 4.3-4.5 (5H, m), 4.5-4.7 (3H, m), 4.97 (1H,d, J=3 Hz), 5.06 (1H, s), 5.20 (1H, d, J=3 Hz), 5.40 (1H, d, J=3 Hz),6.85 (1H, d, J=8.3 Hz), 6.95 (2H, d, J=8.5 Hz), 7.02 (1H, d, J=8.3 Hz),7.30 (1H, d, J=8.5 Hz), 7.44 (1H, s)

FAB-MS: e/z=1259 (M+K)

EXAMPLE 5

FR138364 substance was obtained by reacting FR133303 substance with O⁴-octyl-N-(t-butoxycarbonyl)-L-tyrosine succinimido ester according tothe procedure of Example 3.

IR (Nujol) : 3300, 1660, 1620, 1240, 1050 cm⁻¹

NMR (CD₃ OD, δ): 0.904 (3H, t, J=6.8 Hz), 1.06 (3H, d, J=6.8 Hz), 1.17(3H, d, J=6.7 Hz), 1.20-1.30 (10H, m), 1.35 (9H, s), 1.74 (2H, quintet,J=6.5 Hz), 1.9-2.1 (3H, m), 2.45 (3H, m), 2.76 (1H, dd, J=16 and 4 Hz),3.0-3.1 (2H, m), 3.37 (1H, m), 3.77 (1H, d, J=11 Hz), 3.92 (2H, t, J=6.8Hz), 3.9-4.2 (7H, m), 4.3-4.5 (5H, m), 4.5-4.6 (3H, m), 4.94 (1H, d, J=3Hz), 5.05 (1H, d, J=3.8 Hz), 5.31 (1H, d, J=3 Hz), 6.79 (2H, d, J=8.5Hz), 6.85 (1H, d, J=8.3 Hz), 7.03 (1H, dd, J=8.3 and 2 Hz), 7.12 (2H, d,J=8.5 Hz), 7.31 (1H, d, J=2 Hz)

FAB-MS: e/z=1357 (M+Na)

EXAMPLE 6

A solution of FR133303 substance (0.5 g) in a mixture of water (5 ml)and tetrahydrofuran (5 ml) was adjusted to pH 7 with saturated aqueoussodium bicarbonate, and N,N-di-t-butylcarbonate (0.114 g) was addedthereto at room temperature. The mixture was stirred for 5 hours at roomtemperature, maintaining pH 7 with saturated aqueous sodium bicarbonate.The reaction mixture was added to water and adjusted to pH 6. Theaqueous solution was washed with ethyl acetate, and subjected to ionexchange chromatography an DEAE-TOYOPEARL (Cl.sup.θ) (30 ml), elutingwith 50% methanol in 1M aqueous sodium chloride. The fractionscontaining the object compound were combined and evaporated underreduced pressure to remove methanol. The aqueous solution was adjustedto pH 4.5 with 1N hydrochloric acid and subjected to columnchromatography on DIAION HP-20 (100 ml), eluting with 80% aqueousmethanol. The fractions containing the object compound were combined andevaporated under reduced pressure to remove methanol. The residue waslyophilized to give the object acylated compound (hereinafter referredto as FR138261 substance) (0.145 g).

IR (Nujol): 3300, 1660, 1620, 1240, 1050 cm⁻¹

NMR (CD₃ OD, δ): 1.06 (3H, d, J=6.8 Hz), 1.18 (3H, d, J=6.0 Hz), 1.40(9H, s), 1.9-2.1 (3H, m), 2.44 (3H, m), 2.82 (1H, dd, J=16 and 4 Hz),3.37 (1H, m), 3.75 (1H, d, J=11 Hz), 3.89-4 (2H, m), 4.10 (1H, m), 4.15(1H, m), 4.29 (1H, dd, J=6 and 2 Hz), 4.36-4.45 (5H, m), 4.5-4.6 (3H,m), 4.97 (1H, d, J=3 Hz), 5.06 (1H, dd, J=8.2 and 4 Hz), 5.33 (1H, d,J=3 Hz), 6.85 (1H, d, J=8.3 Hz), 7.03 (1H, dd, J=8.3 and 2 Hz), 7.30(1H, d, J=2 Hz), 7.50 (1H, d, J=8.2 Hz)

FAB-MS: e/z=1081 (M+Na)

EXAMPLE 7

FR138363 substance was obtained by reacting FR133303 substance withacetyl chloride according to the procedure of Example 6.

IR (Nujol): 3300, 1620, 1250, 1040 cm⁻¹

NMR (CD₃ OD, δ): 1.06 (3H, d, J=6.8 Hz), 1.20 (3H, d, J=6 Hz), 1.78-2.05(3H, m), 1.96 (3H, s), 2.21-2.54 (3H, m), 2.95 (1H, m), 3.35-3.42 (1H,m), 3.58-4.42 (11H, m), 4.50-5.05 (5H, m), 5.23 (1H, m), 6.88 (1H, d,J=8.3 Hz), 7.05 (1H, dd, J=8.3 and 2 Hz), 7.35 (1H, d, J=2 Hz)

FAB-MS: 1023 (M+Na)

EXAMPLE 8

FR138728 substance was obtained by reacting FR133303 substance with2-bromoacetyl chloride according to the procedure of Example 6.

IR (Nujol): 3300, 1660, 1620, 1500, 1220, 1040 cm⁻¹

NMR (CD₃ OD, δ): 1.06 (3H, d, J=6.9 Hz), 1.17 (3H, d, J=6.1 Hz), 1.9-2.1(3H, m), 2.50 (3H, m), 2.80 (1H, dd, J=16 and 4 Hz), 3.37 (1H, m),3.6-4.0 (5H, m), 4.09 (1H, m), 4.16 (1H, m), 4.29 (1H, dd, J=6 and 2Hz), 4.36-4.45 (5H, m), 4.5-4.7 (3H, m), 4.97 (1H, d, J=3 Hz), 5.04 (1H,dd, J=8.6 and 4 Hz), 5.25 (1H, d, J=3.1 Hz), 6.85 (1H, d, J=8.3 Hz),7.03 (1H, dd, J=8.3 and 2.1 Hz), 7.31 (1H, d, J=2 Hz), 7.52 (1H, d,J=8.6 Hz)

FAB-MS: e/z =1103 (M+Na)

EXAMPLE 9

FR138538 substance was obtained by reacting FR133303 substance withbenzoyl chloride according to the procedure of Example 6.

IR (Nujol): 3300, 1640, 1240 cm⁻¹

NMR (CD₃ OD, δ): 1.05 (3H, d, J=6.8 Hz), 1.18 (3H, d, J=6 Hz), 1.89-2.12(3H, m), 2.31-2.53 (3H, m), 2.75 (1H, dd, J=12 and 4 Hz), 3.38 (1H, m),3.76 (1H, d, J=11 Hz), 3.87-3.98 (1H, m), 4.02-4.18 (2H, m), 4.22-4.32(4H, m), 4.37-4.40 (3H, m), 4.49-4.62 (3H, m), 4.98 (1H, m), 5.02 (1H,m), 5.37 (1H, d, J=3 Hz), 6.85 (1H, d, J=8.3 Hz), 7.04 (1H, dd, J=8.3and 2.1 Hz), 7.11-7.50 (6H, m)

FAB-MS e/z=1101 (M+Na)

EXAMPLE 10

FR138539 substance was obtained by reacting FR133303 substance with2-(2-aminothiazol-4-yl)-2-thoxyiminoacetic acid according to theprocedure of Example 6.

IR (Nujol): 3300, 1650, 1620, 1520, 12600, 1040 cm⁻¹

NMR (CD₃ OD, δ): 1.05 (3H, d, J=6.8 Hz), 1.21 (3H, d, J=5.9 Hz),1.89-2.21 (3H, m), 2.29-2.61-(3H, m), 2.78-2.89 (1H, m), 3.32-3.42 (1H,m), 3.76-3.82 (1H, m), 3.91-4.01 (2H, m), 3.95 (3H, s), 4.13 (1H, m),4.16 (1H, m), 4.24-4.27 (1H, m), 4.32-4.43 (5H, m), 4.46-4.62 (3H, m),4.97-4.99 (1H, m), 5.08 (1H, m), 5.41 (1H, m), 6.79 (1H, s), 6.86 (1H,d, J=8.1 Hz), 7.04 (1H, dd, J=8.1 and 2 Hz), 7.31 (1H, d, J=2 Hz), 7.51(1H, d, J=7 Hz)

FAB-MS: e/z=1143 (M⁺)

EXAMPLE 11

FR138365 substance was obtained by reacting FR133303 substance withtosyl chloride according to the procedure of Example 6.

IR (Nujol): 3300, 1650, 1620, 1260, 1060 cm⁻¹

NMR (CD₃ OD, δ): 0.75 (3H, d, J=6.8 Hz), 1.07 (3H, d, J=6.0 Hz),1.61-1.79 (1H, m), 1.91-2.05 (3H, m), 2.30-2.59 (3H, m), 3.36 (1H, m),3.68 (1H, d, J=11 Hz), 3.81-4.07 (4H, m), 4.22 (1H, m), 4.32-4.40 (5H,m), 4.42-4.60 (3H, m), 4.7 (1H, m), 5.0 (1H, m), 5.42 (1H, d, J=3 Hz),6.85 (1H d, J=8.3 Hz), 7.03 (1H, dd, J=8.3 and 2 Hz), 7.29-7.33 (3H, m),7.75 (1H, d, J=8.3 Hz)

FAB-MS: e/z=1135 (M+Na)

Preparation 11

To a solution of 6-hydroxy-2-naphthoic acid (1 g) in the mixture of 10%aqueous sodium hydroxide (4.25 ml) and dimethylsulfoxide (17 ml) wasadded octyl bromide (0.918 ml). The mixture was stirred for 6 hours at60° C.

The reaction mixture was added to a mixture of water and ethyl acetateand adjusted to pH 3 with conc. hydrochloric acid. The organic layer wasseparated and dried over magnesium sulfate. The magnesium sulfate wasfiltered off, and the filtrate was evaporated under reduced pressure togive 6-octyloxy-2-naphthoic acid (0.91 g).

IR (Nujol): 1670, 1620, 1210 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.2-1.6 (10H, m), 1.78 (2H,m), 4.10 (2H, t, J=6.7 Hz), 7.19 (1H, dd, J=2.3 and 8.8 Hz), 7.36 (1H,d, J=2.3 Hz), 7.83 (1H, d, J=8.8 Hz), 7.97 (2H, d, J=8.8 Hz), 8.52 (1H,s)

Preparation 12

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.703 g)was added to a solution of 6-octyloxy-2-naphthoic acid (0.85 g) and1-hydroxy-1H-benzotriazole (0.382 g) in ethyl acetate (26 ml). Themixture was stirred for two hours at room temperature.

The reaction mixture was added to water and the separated organic layerwas washed with water and aqueous sodium chloride. The organic layer wasthen dried over magnesium sulfate. The magnesium sulfate was filteredoff, and the filtrate was evaporated under reduced pressure to give3-(6-octyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide (0.74 g).

IR (Nujol): 1770, 1740, 1620, 1190, 1020, 740 cm⁻¹

NMR (CDCl₃, δ): 0.90 (3H, t, J=6.8 Hz), 1.2-1.6 (10H, m), 1.89 (2H, m),4.14 (2H, t, J=6.8 Hz), 7.1-7.3 (2H, m), 7.4-7.6 (3H, m), 7.8-8.0 (2H,m), 8.1-8.2 (2H, m), 8.80 (1H, s)

In the following, the structure of the compound of Example 12 is shown(SEQ ID NO: 1): ##STR20##

    ______________________________________                                        Example                                                                              Compound                                                               No.    No.       R                                                            ______________________________________                                        12     FR139687                                                               ______________________________________                                    

EXAMPLE 12

To a solution of FR133303 substance (0.5 g) and1-(6-octyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide (0.271 g) inN,N-dimethylformamide (1.5 ml) was added 4-dimethylaminopyridine (0.0828g). The mixture was stirred for 12 hours at room temperature.

The reaction mixture was added to water and adjusted to pH 6. Theaqueous solution was washed with ethyl acetate, and subjected to ionexchange chromatography on DEAE-TOYOPEARL (Cl⁻) (30 ml) and eluted with50% methanol in 1M sodium chloride solution. The fractions containingthe object compound were combined and evaporated under reduced pressureto remove methanol. The aqueous solution was adjusted to pH 4.5 with 1 Nhydrochloric acid and subjected to column chromatography on DIAION HP-20(65 ml), eluting with 80% aqueous methanol. The fractions containing theobject compound were combined and evaporated under reduced pressure toremove methanol. The residue was lyophilized to give the object acylatedcompound (hereinafter referred to as FR139687 substance) (0.214 g).

IR (Nujol): 3300, 1620, 1500 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.86 (3H, t, J=6.8 Hz), 0.97 (3H, d, J=6.8 Hz),1.06 (3H, d, J=6.8 Hz), 1.21.5 (10H, m), 1.6 2.0 (5H, m), 2.2-2.5 (3H,m), 2.4-2.6 (1H, m), 3.18 (1H, m), 3.6-3.9 1H, m), 4.0-4.6 (15H, m),4.84 (1H, d, J=3 Hz), 4.90 (1H, d, J=3 Hz), 5.11 (1H, d, J=3 Hz), 6.76(1H, d, J=8.3 Hz), 6.93 (1H, d, J=8.3 Hz), 7.13 (1H, s), 7.25 (1H, d,J=8.3 Hz), 7.39 (1H,,s), 7.8-8.0 (3H, m), 8.44 (1H, s)

FAB-MS e/z=1264 (M+Na)

The following compounds (Preparations 13 to 16) were obtained accordingto methods similar to that of Preparation 5.

Preparation 13

N-(t-Butoxycarbonyl)-L-2-(2-naphthyl)glycine succinimido ester

IR (Nujol): 3350, 1800, 1770, 1730, 1680, 1500, 1200 cm⁻¹

Preparation 14

Succinimido 2-(4-biphenylyl)acetate

IR (Nujol): 1800, 1770, 1720, 1200 cm⁻¹

NMR (DMSO-d₆, δ): 2.82 (4H, s), 4.17 (2H, s), 7.30-7.50 (5H, m), 7.45(2H, d, J=8.1 Hz), 7.67 (2H, d, J=8.1 Hz)

Preparation 15

Succinimido 4-t-butylbenzoate

IR (Nujol): 1760, 1730, 1200, 1070, 990 cm⁻¹

NMR (DMSO-d₆, δ): 1.33 (9H, s), 2.89 (4H, s), 7.68 (2H, d, J=8.5 Hz),8.03 (2H, d, J=8.5 Hz)

Preparation 16

Succinimido 4- (4-phenylbutoxy)benzoate

IR (Nujol): 1730, 1600, 1240, 1170, 1070 cm⁻¹

NMR (DMSO-d₆, δ): 1.75 (4H, m), 2.65 (2H, m), 4.14 (2H, m), 7.15 (2H, d,J=8.9 Hz), 7.13-7.35 (5H, m), 8.03 (2H, d, J=8.9 Hz)

Preparation 17

To neat 3,7-dimethyloctanol (5 ml) was added phosphorus tribromide (1.01ml). The mixture was stirred for 4 hours at 60° C. The reaction mixturewas added to a mixture of water and n-hexane. The organic layer wasseparated and dried over magnesium sulfate. The magnesium sulfate wasfiltered off, and the filtrate was evaporated under reduced pressure togive 3,7-dimethyloctyl bromide (4.40 g).

IR (Neat): 2900, 1450 cm⁻¹

NMR (CDCl₃, δ): 0.87 (6H, d, J=6.6 Hz), 0.89 (3H, d, J=6.4 Hz), 1.1-1.3(6H, m), 1.5-1.9 (4H, m), 3.3-3.5 (2H, m)

The following compounds (Preparations 18 to 23) were obtained accordingto methods similar to that of Preparation 11.

Preparation 18

4-[4-(Octyloxy) phenoxy]benzoic acid

IR (Nujol): 1680, 1600, 1240, 840 cm⁻¹

NMR (DMSO-d₆, δ): 0.87 (3H, t, J=6.7 Hz), 1.1-1.6 (10H, m), 1.71 (2H,m), 3.96 (2H, t, J=6.4 Hz), 6.9-7.1 (6H, m), 7.92 (2H, d, J=8.7 Hz),12.8 (1H, br s)

Preparation 19

6-(Butoxy)-2-naphthoic acid

IR (Nujol): 1660, 1610, 1205 cm⁻¹

NMR (DMSO-d₆, δ): 0.96 (3H, t, J=7.29 Hz), 1.48 (2H, qt, J=7.29 and 7Hz), 1.78 (2H, tt, J=7 and 6.45 Hz), 4.12 (2H, t, J=6.45 Hz), 7.24 (1H,dd, J=9.0 and 2.3 Hz), 7.40 (1H, d, J=2.3 Hz), 7.86 (1H, d, J=8.7 Hz),7.94 (1H, d, J=8.7 Hz), 8.01 (1H, d, J=9.0 Hz), 8.52 (1H, s)

Preparation 20

6-Decyloxy-2-naphthoic acid

IR (Nujol): 1670, 1620, 1210 cm⁻¹

NMR (DMSO-d₆, δ): 0.85 (3H, t, J=6.7 Hz), 1.2-1.6 (14H, m), 1.78 (2H,m), 4.11 (2H, t, J=6.4 Hz), 7.23 (1H, dd, J=8.9 and 2.4 Hz), 7.39 (1H,d, J=2.4 Hz), 7.86 (1H, d, J=8.7 Hz), 7.93 (1H, d, J=8.7 Hz), 8.01 (1H,d, J=8.9 Hz), 8.5 (1H, s)

Preparation 21

6-Hexyloxy-2-naphthoic acid

IR (Nujol): 1660, 1620, 1290, 1210 cm³¹ 1

NMR (DMSO-d₆, δ): 0.89 (3H, t, J=6.8 Hz), 1.2-1.6 (6H, m), 1.78 (2H,quintet, J=6.5 Hz), 4.11 (2H, t, J=6.5 Hz), 7.23 (1H, dd, J=9.0 Hz and2.4 Hz), 7.39 (1H, d, J=2.4 Hz), 7.86 (1H, d, J=8.7 Hz), 7.94 (1H, d,J=8.7 Hz), 8.01 (1H, d, J=9.0 Hz), 8.52 (1H, s)

Preparation 22

6-Dodecyloxy-2-naphthoic acid

IR (Nujol): 1670, 1620, 1210 cm⁻¹

NMR (DMSO-d₆, δ): 0.85 (3H, t, J=6.7 Hz), 1.20-1.60 (18H, m), 1.78 (2H,m), 4.11 (2H, t, J=6.5 Hz), 7.22 (1H, dd, J=9.0 and 2.4 Hz), 7.39 (1H,d, J=2.4 Hz), 7.85 (1H, d, J=8.7 Hz), 7.93 (1H, d, J=8.7 Hz), 8.00 (1H,d, J=9.0 Hz), 8.51 (1H, S), 12.90 (1H, s)

Preparation 23

6-(3,7-Dimethyloctyloxy)-2-naphthoic acid

IR (Nujol): 1660, 1610, 1290, 1210 cm⁻¹

NMR (DMSO-d₆, δ): 0.84 (6H, d, J=6.6 Hz), 0.94 (3H, d, J=6.1 Hz),1.1-1.4 (6H, m), 1.4-1.9 (4H, m), 4.15 (2H, t, J=6.7 Hz), 7.22 (1H, dd,J=9.0 and 2.4 Hz), 7.41 (1H, d, J=2.4 Hz), 7.86 (1H, d, J=8.6 Hz), 7.93(1H, d, J=8.6 Hz), 8.01 (1H, d, J=9.0 Hz), 8.52 (1H, s)

The following compounds (Preparations 24 to 31) were obtained accordingto methods similar to that of Preparation 12.

Preparation 24

1-[4-(4-octyloxy)phenoxy]benzoyl-1H-benzotriazole-3-oxide

IR (Nujol): 1770, 1730, 1600, 1500, 1230, 980 cm⁻¹

Preparation 25

1-(6-Butoxy-2-naphthoyl)-1H-benzotriazole-3-oxide

IR (Nujol): 1760, 1610, 1260, 1180, 1020 cm⁻¹

Preparation 26

1-(6-Decyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide

IR (Nujol): 1780, 1620, 1190, 1000 cm⁻¹

Preparation 27

1-(6-Hexyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide

IR (Nujol): 1780, 1610, 1190 cm⁻¹

NMR (DMSO-d₆, δ): 0.89 (3H, t, J=6.7 Hz), 1.2-1.6 (6H, m), 1.79 (2H, m),4.12 (2H, t, J=6.5 Hz), 7.24 (1H, dd, J=9.0 and 2.4 Hz), 7.39 (1H, d,J=2.4 Hz), 7.41 (1H, t, J=8 Hz), 7.54 (1H, t, J=8 Hz), 7.72 (1H, d, J=8Hz), 7.88 (1H, d, J=8.7 Hz), 7.90 (1H, d, J=8.7 Hz), 7.97 (1H, d, J=8Hz), 8.02 (1H, d, J=9.0 Hz), 8.51 (1H, s)

Preparation 28

1-(6-Dodecyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide

IR (Nujol): 1770, 1620, 1190, 1030, 730 cm⁻¹

NMR (DMSO-d₆, δ): 0.85 (3H, t, J=6.7 Hz), 1.2-1.3 (18H, m), 1.78 (2H,m), 4.11 (2H, t, J=6.5 Hz), 7.22 (1H, dd, J=9.0 and 2.4 Hz), 7.39 (1H,d, J=2.4 Hz), 7.40 (1H, t, J=8 Hz), 7.55 (1H, t, J=8 Hz), 7.73 (1H, d,J=8 Hz), 7.85 (1H, d, J=8.7 Hz), 7.93 (1H, d, J=8.7 Hz), 7.99 (1H, d,J=8 Hz), 8.00 (1H, d, J=9.0 Hz), 8.51 (1H, s)

Preparation 29

1-[6-(3,7-Dimethyloctyloxy)-2-naphthoyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1780, 1620, 1190 cm⁻¹

Preparation 30

1-[(2E,6E)-3,7,11-Trimethyl-2,6,10-dodecatrienoyl]-1H-benzotriazole-3-oxide

IR (Neat): 2900, 1780, 1620, 1420, 1070 cm⁻¹

Preparation 31

3,7-Dimethyl-6-octenyl bromide was obtained according to the method ofPreparation 17.

IR (Neat): 2900, 1440, 1380 cm^(-l)

NMR (DMSO-d₆, δ): 0.86 (3H, d, J=6.3 Hz), 1.0-1.5 (2H, m), 1.57 (3H, s),1.65 (3H, s), 1.7-2.1 (5H, m), 3.4-3.7 (2H, m), 5.08 (1H, m)

Preparation 32

To a suspension of sodium hydride (2.04 g) in N,N-dimethylformamide (50ml) was added 4-hydroxypyridine (5 g) at room temperature. Octyl bromide(9.08 ml) was added thereto. The mixture was stirred for 2 hours at 50°C. The reaction mixture was added to a mixture of brine (100 ml),tetrahydrofuran (100 ml) and ethyl acetate (100 ml). The organic layerwas separated and dried over magnesium sulfate. The magnesium sulfatewas filtered off, and the filtrate was evaporated under reduced pressureto give 1-octyl-4-pyridone (14.7 g).

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6 Hz), 1.1-1.4 (10H, m), 1.4-1.8 (2H,m), 3.81 (2H, t, J=7 Hz), 6.05 (2H, d, J=8 Hz), 7.63 (2H, d, J=8 Hz)

Preparation 33

To a solution of 1-octyl-4-pyridone (10.9 g) in pyridine (100 ml) wasadded phosphorous pentasulfide (8.65 g) at room temperature. The mixturewas stirred for 3 hours at 80° C. The reaction mixture was added to amixture of water (200 ml) and methylene chloride (200 ml). The organiclayer was separated and dried over magnesium sulfate. The magnesiumsulfate was filtered off, and the filtrate was evaporated under reducedpressure to give 1-octyl-1,4-dihydropyridine-4-thione (5.27 g).

IR (Neat): 2910, 2850, 1620, 1460, 1110 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6 Hz), 1.1-1.4 (10H, m), 1.5-1.9 (2H,m), 3.95 (2H, t, J=7 Hz), 7.13 (2H, d, J=7 Hz), 7.60 (2H, d, J=7 Hz)

The following compounds (Preparations 34 to 36) were obtained accordingto methods similar to that of Preparation 1.

Preparation 34

Methyl 2-(4 -hydroxyphenyl)-2-methoxyacetate

IR (Nujol): 3350, 1740, 1610, 1600, 1220, 1100 cm⁻¹

NMR (DMSO-d₆, δ): 3.23 (3H, s) , 3.60 (3H, s) , 4.73 (1H, s), 6.72 (2H,d, J=8.9 Hz), 7.15 (2H, d, J=8.9 Hz)

EI-MS (e/z)=196 (M⁺)

Preparation 35

D-Tyrosine methyl ester hydrochloride

IR (Nujol): 3300, 1740, 1220 cm⁻¹

NMR (DMSO-d₆, δ): 3.02 (2H, m), 3.67 (3H, s), 4.16 (1H, t, J=6.7 Hz),6.72 (2H, d, J=8.4 Hz), 7.01 (2H, d, J=8.4 Hz), 8.58 (2H, s), 9.47 (1H,s)

Preparation 36

Methyl (4 -hydroxyphenyl)glyoxylate

IR (Nujol): 3380, 1730, 1700, 1600, 1580, 1220 cm⁻¹

NMR (DMSO-d₆, δ): 3.91 (3H, s), 6.94 (2H, d, J=8.8 Hz), 7.83 (2H, d,J=8.8 Hz), 10.9 (1H, s)

Preparation 37

N-(t-Butoxycarbonyl)-D-tyrosine methyl ester was obtained according tothe method of Preparation 2.

IR (Nujol): 3360, 1700, 1680, 1290, 1270, 1250 cm⁻¹

NMR (DMSO-d₆, δ): 1.33 (9H, s), 2.73 (2H, m), 3.59 (3H, s), 4.05 (1H,m), 6.65 (2H, d, J=8.4 Hz), 7.00 (2H, d, J=8.4 Hz), 7.23 (1H, d, J=7.9Hz), 9.23 (1H, s)

Preparation 38

To a solution of L-tyrosine methyl ester hydrochloride (1 g) in water(1.5 ml) was added sodium bicarbonate (0.363 g) under ice-cooling. Themixture was stirred for 10 minutes, and then acetonitrile (7 ml), 37%aqueous formaldehyde (0.637 ml) and sodium cyanoborohydride (0.182 g)were added thereto at -5° C. The mixture was stirred for 2 hours at -5°C. The resultant insoluble material was filtered off, and the filtratewas extracted with ethyl acetate. The organic layer was separated anddried over magnesium sulfate. The magnesium sulfate was filtered off,and the filtrate was evaporated under reduced pressure to giveN,N-dimethyl-L-tyrosine methyl ester (0.21 g) .

IR (Nujol): 1730, 1260, 1010 cm⁻¹

NMR (DMSO-d₆, δ): 2.24 (6H, s), 2.72 (2H, m), 3.34 (1H, m), 3.53 (3H,s), 6.64 (2H, d, J=8.4 Hz), 6.97 (2H, d, J=8.4 Hz), 9.18 (1H, s)

The following compounds (Preparations 39 to 44) were obtained accordingto methods similar to that of Preparation 3.

Preparation 39

Methyl 2-(4 -octyloxyphenyl)acetate

IR (Neat): 2910, 2850, 1730, 1240 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.3 Hz), 1.2-1.5 (10H, m), 1.6-1.9 (2H,m), 3.58 (2H, s), 3.59 (3H, s), 3.92 (2H, t, J=6.4 Hz), 6.85 (2H, d,J=8.7 Hz), 7.15 (2H, d, J=8.7 Hz)

Preparation 40

Ethyl 3-(4-octyloxyphenyl)propionate

IR (Neat): 2920, 2850, 1730, 1240 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.15 (3H, t, J=7.1 Hz),1.2-1.5 (10H, m), 1.6-1.8 (2H, m), 2.55 (2H, t, J=7.2 Hz), 2.77 (2H, t,J=7.2 Hz), 3.90 (2H, t, J=6.4 Hz), 4.03 (2H, q, J=7.1 Hz), 6.81 (2H, d,J=8.6 Hz), 7.11 (2H, d, J=8.6 Hz)

Preparation 41

Methyl 2-(4-octyloxyphenyl)-2-methoxyacetate

IR (Neat): 2910, 2850, 1740, 1600, 1240, 1100 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.8 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 3.26 (3H, s), 3.62 (3H, s), 3.94 (2H, t, J=6.4 Hz), 4.83 (1H, s),6.91 (2H, d, J=8.7 Hz), 7.27 (2H, d, J=8.7 Hz)

EI-MS (e/z)=308 (M⁺)

Preparation 42

O⁴ -Octyl-N-(t-butoxycarbonyl)-D-tyrosine methyl ester

IR (Nujol): 3350, 1730, 1680, 1510, 1240, 1160 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.2-1.3 (10H, m), 1.68 (2H,m), 2.82 (2H, m), 3.60 (3H, s), 3.91 (2H, t, J=7.3 Hz), 4.08 (1H, m),6.81 (2H, d, J=8.6 Hz), 7.12 (2H, d, J=8.6 Hz), 7.25 (1H, d, J=8.0 Hz)

Preparation 43

O⁴ -Octyl-N,N-dimethyl-L-tyrosine methyl ester

IR (Neat): 2930, 2860, 1730, 1250 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.6 Hz), 1.26 (10H, m), 1.68 (2H, m),2.80 (2H, m), 3.33 (6H, s), 3.37 (1H, m), 3.53 (3H, s), 3.89 (2H, t,J=6.4 Hz), 6.79 (2H, d, J=8.6 Hz) , 7.08 (2H, d, J=8.6 Hz)

Preparation 44

Methyl (4-octyloxyphenyl)glyoxylate

IR (Neat): 2930, 2850, 1730, 1670, 1600, 1260, 1210, 1160 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.3 Hz), 1.2-1.5 (10H, m), 1.6-1.9 (2H,m), 3.93 (3H, s), 4.10 (2H, t, J=6.5 Hz), 7.12 (2H, d, J=8.9 Hz), 7.92(2H, d, J=8.9 Hz)

The following compounds (Preparations 45 to 51) were obtained accordingto methods similar to that of Preparation 4.

Preparation 45

4-(2-Butoxyethoxy)benzoic acid

IR (Nujol): 1670, 1610, 1260 cm⁻¹

NMR (DMSO-d₆, δ): 0.87 (3H, t, J=7.2 Hz), 1.2-1.6 (4H, m), 3.45 (2H, t,J=6.4 Hz), 3.70 (2H, t, J=4.4 Hz), 4.16 (2H, t, J=4.4 Hz), 7.02 (2H, d,J=8.9 Hz), 7.88 (2H, d, J=8.9 Hz), 12.63 (1H, s)

Preparation 46

2-(4-Octyloxyphenyl)acetic acid

IR (Nujol): 1680, 1240, 820, 780 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.8 Hz), 1.1-1.5 (10H, m), 1.6-1.8 (2H,m), 3.47 (2H, s), 3.92 (2H, t, J=6.4 Hz), 6.84 (2H, d, J=8.6 Hz), 7.14(2H, d, J=8.6 Hz)

Preparation 47

3-(4-Octyloxyphenyl)propionic acid

IR (Nujol): 1680, 1500, 1200 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.3 Hz), 1.1-1.5 (10H, m), 1.6-1.8 (2H,m), 2.47 (2H, t, J=7.2 Hz), 2.74 (2H, t, J=7.2 Hz), 3.90 (2H, t, J=6.4Hz), 6.81 (2H, d, J=8.6 Hz), 7.11 (2H, d, J=8.6 Hz), 12.10 (1H, br s)

Preparation 48

2-(4-Octyloxyphenyl)-2-methoxyacetic acid

IR (Nujol): 1760, 1720, 1600, 1500, 1240, 1180, 1100, 830 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.2-1.5 (10H, m), 2.6-2.8 (2H,m), 3.26 (3H, s), 3.94 (2H, t, J=6.4 Hz), 4.67 (1H, s), 6.90 (2H, d,J=8.6 Hz), 7.27 (2H, d, J=8.6 Hz)

Preparation 49

O⁴ -Octyl-N-(t-butoxycarbonyl)-D-tyrosine

IR (Nujol): 3400-2900, 1700, 1500, 1240, 1160 cm⁻¹

NMR (DMSO-d₆, δ): 0.859 (3H, t, J=6.8 Hz), 1.20-1.30 (10H, m), 1.32 (9H,s), 1.68 (2H, m), 2.67-2.95 (1H, m), 3.90 (2H, t, J=7 Hz), 4.01 (1H, m),6.81 (2H, d, J=8.6 Hz), 7.02 (1H, d, J=8.3 Hz), 7.13 (2H, d, J=8.6 Hz)

Preparation 50

O⁴ -Octyl-N,N-dimethyl-L-tyrosine

IR (Neat): 2940, 2860, 2600, 1620, 1240 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.6 Hz), 1.26 (10H, m), 1.68 (2H, m),2.67 (6H, s), 2.8-3.6 (3H, m), 3.91 (2H, t, J=6.4 Hz), 6.85 (2H, d,J=8.5 Hz), 7.16 (2H, d, J=8.5 Hz)

Preparation 51

O⁴ -Octyloxyphenylglyoxylic acid

IR (Neat): 1730, 1670, 1600, 1260, 1160 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.8 Hz), 1.2-1.5 (10H, m), 1.65-1.85(2H, m), 4.09 (2H, t, J=6.5 Hz), 7.12 (2H, d, J=8.9 Hz), 7.89 (2H, d,J=8.9 Hz)

Preparation 52

N.sup.τ -Octyl-N-(t-butoxycarbonyl)-L-histidine was obtained fromN-(t-butoxycarbonyl)-L-histidine methyl ester according to a proceduresimilar to those of Preparations 3 and 4.

NMR (DMSO-d₆, δ): 0.85 (3H, t, J=6.3 Hz), 1.23 (10H, m), 1.35 (9H, s),2.83 (2H, m), 3.90 (2H, t, J=7 Hz), 4.0-4.2 (1H, m), 6.36 (1H, s), 7.02(1H, d, J=8 Hz), 7.75 (1H, s)

The following compounds (Preparations 53 to 60) were obtained accordingto procedures similar to that of Preparation 11.

Preparation 53

4-Octyloxyphthalic acid

IR (Neat): 2930, 2860, 2500, 1700, 1600, 1260 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.8 Hz), 1.2-1.5 (10H, m), 1.5-1.8 (2H,m), 4.05 (2H, t, J=6.2 Hz), 7.03 (1H, d, J=2.6 Hz), 7.06 (1H, dd, J=8.4and 2.6 Hz), 7.72 (1H, d, J=8.4 Hz)

Preparation 54

3-Methoxy-4-octyloxybenzoic acid

IR (Nujol): 2600, 1680, 1600, 1270, 1230 cm⁻¹

NMR (DMSO-d₆, δ) 0.86 (3H, t, J=6.8 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 3.80 (3H, s), 4.01 (2H, t, J=6.5 Hz), 7.03 (1H, d, J=8.5 Hz), 7.44(1H, d, J=1.9 Hz), 7.54 (1H, dd, J=8.5 and 1.9 Hz)

Preparation 55

4-(4-Octyloxyphenyl)benzoic acid

IR (Nujol): 1670, 1600, 830, 770 cm⁻¹

NMR (DMSO-d₆, δ): 0.87 (3H, t, J=6.7 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 4.01 (2H, t, J=6.4 Hz), 7.04 (2H, d, J=8.8 Hz), 7.68 (2H, d, J=8.8Hz), 7.75 (2H, d, J=8.5 Hz), 7.99 (2H, d, J=8.5 Hz)

Preparation 56

6-(2 -Ethylhexyloxy)-2-naphthoic acid

IR (Nujol): 1660, 1610, 1280, 1200 cm⁻¹

NMR (DMSO-d₆, δ): 0.88 (3H, t, J=7.3 Hz), 0.92 (3H, t, J=7.3 Hz),1.2-1.6 (8H, m), 1.7-1.9 (1H, m), 4.01 (2H, d, J=5.7 Hz), 7.23 (1H, dd,J=8.9 and 2.4 Hz), 7.42 (1H, d, J=2.4 Hz), 7.86 (1H, d, J=8.7 Hz), 7.94(1H, d, J=8.7 Hz), 8.01 (1H, d, J=8.9 Hz), 8.51 (1H, s), 12.9 (1H, s)

Preparation 57

6-(3,7-Dimethyl-6-octenyloxy)naphthoic acid

IR (Nujol): 1660, 1610, 1290, 1200 cm⁻¹

NMR (DMSO-d₆, δ): 0.95 (3H, d, J=6.1 Hz), 1.1-1.5 (2H, m), 1.57 (3H, s),1.64 (3H, s), 1.6-2.1 (5H, m), 4.15 (2H, t, J=6.7 Hz), 5.10 (1H, t,J=7.1 Hz), 7.22 (1H, dd, J=8.9 and 2.3 Hz), 7.42 (1H, d, J=2.3 Hz), 7.86(1H, d, J=8.6 Hz), 7.94 (1H, d, J=8.6 Hz), 8.01 (1H, d, J=8.9 Hz), 8.52(1H, s), 12.89 (1H, s)

Preparation 58

6-(3,7-Dimethyl-2,6-octadienyloxy)naphthoic acid

IR (Nujol): 1660, 1620, 1210 cm⁻¹

NMR (DMSO-d₆, δ): 1.57 (3H, s), 1.60 (3H, s), 1.76 (3H, s), 2.07 (4H,m), 4.70 (2H, d, J=6.5 Hz), 5.07 (1H, m), 5.51 (1H, t, J=6.5 Hz), 7.24(1H, dd, J=8.9 and 2.4 Hz), 7.41 (1H, d, J=2.4 Hz), 7.85 (1H, d, J=8.7Hz), 7.94 (1H, d, J=8.7 Hz), 8.01 (1H, d, J=8.9 Hz), 8.52 (1H, s), 12.88(1H, s)

Preparation 59

(2E)-3-(4-Octyloxyphenyl)acrylic acid

IR (Nujol): 1660, 1600, 1240 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 4.00 (2H, t, J=6.4 Hz), 6.36 (1H, d, J=16 Hz), 6.95 (2H, d, J=8.7Hz), 7.54 (1H, d, J=16 Hz), 7.61 (2H, d, J=8.7 Hz), 12.20 (1H, br s)

Preparation 60

Sodium 6-octyloxy-2-naphthalene sulfonate

IR (Nujol): 1230, 1180, 860, 820 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6 Hz), 1.1-1.6 (10H, m), 4.06 (2H, t,J=5 Hz), 7.08 (1H, d, J=9 Hz), 7.21 (1H, s), 7.79 (1H, d, J=9 Hz), 8.00(1H, s)

Preparation 61

To a solution of thionyl chloride (0.692 ml) and N,N-dimethylformamide(0.022 ml) was added sodium 6-octyloxy-2-naphthalenesulfonate (1 g)under ice-cooling. The mixture was stirred for 1.5 hours at 95° C., thenevaporated under reduced pressure to give 6-octyloxy-2-naphthylsulfonylchloride (1 g) .

IR (Nujol): 1610, 1260, 1160 cm⁻¹

NMR (CDCl₃, δ): 0.90 (3H, t, J=6.2 Hz), 1.2-1.7 (10H, m), 1.8-2.0 (2H,m), 4.12 (2H, t, J=6.5 Hz), 7.20 (1H, d, J=2.2 Hz), 7.32 (1H, dd, J=9.0and 2.2 Hz), 7.84-7.97 (3H, m), 8.49 (1H, s)

The following compounds (Preparations 62 to 71) were obtained accordingto procedures similar to that of Preparation 12.

Preparation 62

1-(4-Octylbenzoyl)-1H-benzotriazole-3-oxide

IR (Neat): 2930, 2850, 1780, 1610, 1240, 990 cm⁻¹

Preparation 63

1-[4-(4-Octyloxyphenyl)benzoyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1770, 1600, 980 cm⁻¹

Preparation 64

1-[6-(2-Ethylhexyloxy)-2-naphthoyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1770, 1620, 1270, 1180 cm⁻¹

NMR (CDCl₃, δ): 0.93 (3H, t, J=7.1 Hz), 0.98 (3H, t, J=7.4 Hz), 1.3-1.7(8H, m), 1.7-2.0 (1H, m), 4.03 (2H, d, J=5.7 Hz), 7.22 (1H, d, J=2.2Hz), 7.29 (1H, dd, J=8.9 and 2.2 Hz), 7.4-7.7 (3H, m), 7.87 (1H, d,J=9.5 Hz), 7.92 (1H, d, J=9.5 Hz) , 8.1-8.2 (2H, m), 8.80 (1H, s)

Preparation 65

1-[6-(3,7-Dimethyl-6-octenyloxy)-2-naphthoyl]-1H-benzotriazole-3-oxide

IR (Neat): 2900, 1770, 1620, 1180 cm⁻¹

Preparation 66

1-[6-{(E)-3,7-Dimethyl-2,6-octadienyloxy}-2-naphthoyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1770, 1620, 1270, 1180 cm⁻¹

Preparation 67

1-(2-Anthrylcarbonyl)-1H-benzotriazole-3-oxide

IR (Nujol): 1780, 1200, 720, 740 cm⁻¹

Preparation 68

1-[2-(4-Octyloxyphenyl)acetyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1730, 1460, 1420, 1250, 1130 cm⁻¹

Preparation 69

1-[3-(4-Octyloxyphenyl)propionyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1730, 1420, 1340, 1240, 950 cm⁻¹

Preparation 70

1-[(E)-3-(4-Octyloxyphenyl)acryloyl]-1H-benzotriazole-3-oxide

IR (Nujol): 1770, 1600, 1260, 1080 cm⁻¹

Preparation 71

1-(O⁴ -Octyl-N,N-dimethyl-L-tyrosyl)-1H-benzotriazole-3-oxide

IR (Neat): 2930, 2850, 1800, 1610 cm⁻¹

Preparation 72

To a suspension of lithium aluminum hydride (4.05 g) in tetrahydrofuran(475 ml) was added dropwise a solution of 4-octyloxybenzaldehyde (25 g)in tetrahydrofuran (25 ml) at 55°-60° C. The reaction mixture wasstirred under reflux for 1 hour. Thereto, sodium fluoride (35.84 g) andwater (11.52 ml) were added under ice-cooling. The mixture was stirredfor 30 minutes, and filtered. The filtrate was evaporated in vacuo togive 4-octyloxybenzyl alcohol (25.1 g) as crystals.

IR (Nujol): 3200, 1605, 1510 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.26-1.38 (10H, m), 1.62-1.72(2H, m), 3.92 (2H, t, J=6.5 Hz), 4.40 (2H, d, J=5.7 Hz), 5.03 (1H, t,J=5.7 Hz), 6.85 (2H, d, J=8.6 Hz), 7.20 (2H, d, J=8.6 Hz)

Preparation 73

Diethyl azodicarboxylate (18.4 g) was added dropwise to a suspension of4-octyloxybenzyl alcohol (25 g), N-hydroxyphthalimide (17.15 g) andtriphenylphosphine (27.74 g) in tetrahydrofuran (250 ml) underice-cooling. The reaction mixture was stirred at room temperature for 2hours, and evaporated in vacuo. The residue was purified bychromatography on silica gel to give N-(4-octyloxybenzyloxy)phthalimide(33.45 g) as crystals.

IR (Nujol): 1780, 1725, 1605, 1580, 1505 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, m), 1.26 (10H, m), 1.70 (2H, m), 3.95 (2H,t, J=6.5 Hz), 5.08 (2H, s), 6.93 (2H, d, J=8.6 Hz), 7.40 (2H, d, J=8.6Hz), 7.85 (4H, s)

Preparation 74

To a solution of N-(4-octyloxybenzoyloxy)phthalimide (4.13 g) intetrahydrofuran (16 ml) was added hydrazine hydrate (0.53 ml) at roomtemperature. After the mixture was stirred at the same temperature for 1hour, the precipitate was filtered off. To the filtrate were added water(6 ml) and 4-hydroxyphenylglyoxylic acid (1.5 g) at room temperature.The mixture was maintained at pH 4-4.5 with aqueous sodium bicarbonatesolution for 2 hours. Thereto was added ethyl acetate, and adjusted topH 2 with 1N hydrochloric acid. The separated organic layer was washedwith brine, and dried over magnesium sulfate. The magnesium sulfate wasfiltered off, and the organic solvent was evaporated in vacuo to give2-(4 -hydroxyphenyl)-2-(4 -octyloxybenzyloxyimino) acetic acid (3.4 g).

IR (Nujol): 3400, 1715, 1605, 1590, 1505 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, m), 1.25 (10H, m), .1.69 (2H, m), 3.94 (2H,t, J=6.4 Hz), 5.07 (2H, s), 6.82 (2H, d, J=8.7 Hz), 6.90 (2H, d, J=8.6Hz), 7.29 (2H, d, J=8.6 Hz), 7.35 (2H, d, J=8.7 Hz)

The following compounds (Preparations 75 and 76) were obtained accordingto procedures similar to that of Preparation 74.

Preparation 75

2-Phenyl-2-(4-octyloxybenzyloxyimino)acetic acid

IR (Nujol): 1720, 1610, 1585, 1515 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.26 (10H, m), 1.69 (2H, m),3.94 (2H, t, J=6.5 Hz), 5.13 (2H, s), 6.91 (2H, d, J=8.6 Hz), 7.22-7.49(7H, m)

Preparation 76

2- (4-Octyloxybenzyloxyimino)acetic acid

IR (Nujol): 1700, 1670, 1600 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.2 Hz), 1.26 (10H, m), 1.70 (2H, m),3.95 (2H, t, J=6.5 Hz), 5.13 (2H, s), 6.91 (2H, d, J=8.6 Hz), 7.29 (2H,d, J=8.6 Hz), 7.56 (1H, s)

Preparation 77

A solution of 4-octyloxyphenylglyoxylic acid (0.935 g) in a mixture ofwater (9 ml) and tetrahydrofuran (18 ml) was adjusted to pH 3.5-4 with1N hydrochloric acid, and methoxyamine hydrochloride (0.337 g) was addedthereto at room temperature. The mixture was stirred for 2 hours at roomtemperature maintaining pH 3.5-4 with 1N hydrochloric acid. The reactionmixture was added to ethyl acetate. The organic layer was separated anddried over magnesium sulfate. The magnesium sulfate was filtered off,and the filtrate was evaporated under reduced pressure to give2-(4-octyloxyphenyl)-2-methoxyiminoacetic acid (0.57 g).

IR (Nujol): 1700, 1600, 1250, 1030 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.3 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 3.89 (3H, s), 3.99 (2H, t, J=6.4 Hz), 7.00 (2H, d, J=8.9 Hz), 7.45(2H, d, J=8.9 Hz), 14.05 (1H, s)

Preparation 78

To a mixture of 2,3,4,5,6-pentafluorobenzoic acid (1 g) and2,2,3,3,4,4,5,5-octafluoropentanol (1.18 g) in N,N-dimethylformamide (5ml) was added 62% sodium hydride (0.39 g) at room temperature. Themixture was stirred at the same temperature for 1 hour, and thereto wasadded a mixture of water and ethyl acetate. The separated organic layerwas washed with water and brine, dried over magnesium sulfate, filtered,and evaporated in vacuo. The residue was purified by chromatography onsilica gel to give4-(2,2,3,3,4,4,5,5-octafluoropentyloxy)-2,3,5,6-tetrafluorobenzoic acid(923.0 mg).

IR (Nujol): 1700, 1580 cm⁻¹

NMR (DMSO-d₆, δ): 4.96 (2H, t, J=14.2 Hz), 7.10 (1H, tt,

J=5.6 and 50.2 Hz)

Preparation 79

4-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-Pentadecafluorooctyloxy)-2,3,5,6-tetrafluorobenzoicacid was prepared by a procedure similar to that of Preparation 78.

IR (Nujol): 3400, 1640, 1560 cm⁻¹

NMR (DMSO-d₆, δ): 4.95 (2H, t, J=14.0 Hz)

The following compounds (Preparations 80 to 90) were obtained accordingto procedures similar to that of Preparation 5.

Preparation 80

Succinimido 2-(4-hydroxyphenyl)-2-(4-octyloxybenzyloxyimino)acetate

IR (Nujol): 1800, 1770, 1700, 1600 cm⁻¹

Preparation 81

Succinimido 2-phenyl-2-(4 -octyloxybenzyloxyimino)-acetate

IR (Nujol): 1780, 1730, 1605 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, m), 1.26 (10H, m), 1.69 (2H, m), 2.90 (4H,m), 3.94 (2H, t, J=6.4 Hz), 5.30 (2H, s), 6.91 (2H, d, J=8.6 Hz),7.25-7.56 (7H, m)

Preparation 82

Succinimido 2-(4-Octyloxybenzyloxyimino)acetate

IR (Nujol): 1760, 1725, 1600, 1580 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.26 (10H, m), 1.70 (2H, m),2.85 (4H, s), 3.96 (2H, m), 5.28 (2H, s), 6.91 (2H, d, J=8.6 Hz), 7.33(2H, d, J=8.6 Hz), 8.12 (1H, s)

Preparation 83

Succinimido4-(2,2,3,3,4,4,5,5-octafluoropentyloxy)-2,3,5,6-tetraflurobenzoate

IR (Nujol): 3500, 1770, 1740, 1640 cm⁻¹

NMR (DMSO-d₆, δ): 2.90 (4H, s), 5.23 (2H, t, J=13.8 Hz), 7.11 (1H, tt,J=50.2 and 5.6 Hz)

Preparation 84

Succinimido4-(2,2,3,3,4p4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyloxy)-2,3,5,6-tetrafluorobenzoate

IR (Nujol): 1735, 1620, 1600 cm⁻¹

NMR (DMSO-d₆, δ): 2.90 (4H, s), 5.12 (2H, t, J=13.8 Hz)

Preparation 85

Succinimido 3-methoxy-4-octyloxybenzoate

IR (Nujol): 1760, 1730, 1600, 1280, 1200, 880 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.2-1.5 (10H, m), 1.6-1.9 (2H,m), 2.88 (4H, s), 3.84 (3H, s), 4.09 (2H, t, J=6.5 Hz), 7.19 (1H, d,J=8.6 Hz), 7.49 (1H, d, J=2.0 Hz), 7.73 (1H, dd, J=8.6 and 2.0 Hz)

Preparation 86

Succinimido 4-(2-butoxyethoxy)benzoate

IR (Nujol): 1730, 1600, 1250, 1060 cm⁻¹

NMR (DMSO-d₆, δ): 0.87 (3H, t, J=7.2 Hz), 1.2-1.6 (4H, m), 2.89 (4H, s),3.46 (2H, t, J=6.3 Hz), 3.73 (2H, t, J=4.4 Hz), 4.25 (2H, t, J=4.4 Hz),7.18 (2H, d, J=9.0 Hz), 8.04 (2H, d, J=9.0 Hz)

Preparation 87

Succinimido 2-(4-octyloxyphenyl) -2-methoxyacetate

IR (Nujol): 1810, 1740, 1610, 1250, 1210, 1100 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.7 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 2.80 (4H, s), 3.35 (3H, s), 3.97 (2H, t, J=6.4 Hz), 5.35 (1H, s),6.96 (2H, d, J=8.7 Hz), 7.38 (2H, d, J=8.7 Hz)

Preparation 88

O⁴ -Octyl-N-(t-butoxycarbonyl)-D-tyrosine succinimido ester

IR (Nujol): 3370, 1780, 1730, 1700, 1250, 1200 cm⁻¹

Preparation 89

Succinimido 2-(4-octyloxyphenyl)-2-methoxyiminoacetate

IR (Nujol): 1800, 1780, 1730, 1600, 1250, 1180, 1130 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.6 Hz), 1.2-1.5 (10H, m), 1.6-1.8 (2H,m), 2.89 (4H, s), 4.01 (3H, s), 4.03 (2H, t, J=6.4 Hz), 7.08 (2H, d,J=8.9 Hz), 7.68 (2H, d, J=8.9 Hz)

Preparation 90 N.sup.τ -Octyl-N-(t-butoxycarbonyl)-L-histidinesuccinimido ester

IR (Neat): 1810, 1780, 1730, 1500, 1360, 1200, 1160 cm⁻¹

Preparation 91

4-Octyloxyphthalic anhydride was obtained from 4-octyloxyphthalic acidaccording to a procedure similar to that of Preparation 5.

IR (Neat): 2910, 2850, 1840, 1760, 1640, 1610, 1290, 1260 cm⁻¹

NMR (DMSO-d₆, δ): 0.86 (3H, t, J=6.8 Hz), 1.2-1.5 (10H, m), 1.6-1.9 (2H,m), 4.19 (2H, t, J=6.5 Hz), 7.47 (1H, dd, J=8.4 and 2.2 Hz), 7.57 (1H,d, J=2.2 Hz), 7.98 (1H, d, J=8.4 Hz)

Preparation 92

N-Octyloxycarbonyloxysuccinimide was obtained according to a proceduresimilar to that of Preparation 5.

IR (Neat): 2960, 2850, 1780, 1740, 1260, 1230 cm⁻¹

NMR (CDCl₃, δ): 0.89 (3H, t, J=6.7 Hz), 1.2-1.4 (10H, m), 1.6-1.8 (2H,m), 2.84 (4H, s), 4.32 (2H, t, J=6.7 Hz)

Preparation 93

To a solution of octyl phenyl ether (1.53 g) in chloroform (6 ml) wasadded chlorosulfonic acid at 0° C. The mixture was stirred at roomtemperature for 30 minutes, then the mixture was poured into a mixtureof water and tetrahydrofuran.

The separated organic layer was washed with aqueous sodium chloride,dried over magnesium sulfate and then the solvent was evaporated invacuo. The residue was subjected to a column chromatography on silicagel to give 4-octyloxyphenylsulfonyl chloride (1.25 g).

IR (Nujol): 1600, 1580, 1500, 1380, 1180 cm⁻¹

NMR (CDCl₃, δ): 0.89 (3H, t, J=6.6 Hz), 1.20-1.50 (10H, m), 1.80 (2H,m), 4.06 (2H, t, J=6.4 Hz), 7.03 (2H, d, J=9.0 Hz), 7.96 (2H, d, J=9.0Hz)

In the following Table, the structures of the R groups of the compoundsof Examples 13 to 53 are shown, wherein the compounds have the followinggeneral formula: ##STR21##

In the following formulae, ^(t) Bu means t-butyl, and p-TsOH meansp-toluenesulfonic acid.

    ______________________________________                                        Ex-                                                                           am-  Com-                                                                     ple  pound                                                                    No.  No.      R                                                               ______________________________________                                        13   FR139835 COO(CH.sub.2).sub.7 CH.sub.3                                    14   FR139537                                                                                ##STR22##                                                      15   FR141145                                                                                ##STR23##                                                      16   FR139538                                                                                ##STR24##                                                      17   FR140215                                                                                ##STR25##                                                      18   FR140216                                                                                ##STR26##                                                      19   FR140727                                                                                ##STR27##                                                      20   FR143301                                                                                ##STR28##                                                      21   FR140495                                                                                ##STR29##                                                      22   FR139503                                                                                ##STR30##                                                      23   FR139500                                                                                ##STR31##                                                      24   FR139501                                                                                ##STR32##                                                      25   FR139502                                                                                ##STR33##                                                      26   FR138959                                                                                ##STR34##                                                      27   FR140291                                                                                ##STR35##                                                      28   FR141580                                                                                ##STR36##                                                      29   FR141579                                                                                ##STR37##                                                      30   FR141146                                                                                ##STR38##                                                      31   FR140731                                                                                ##STR39##                                                      32   FR140217                                                                                ##STR40##                                                      33   FR142472                                                                                ##STR41##                                                      34   FR140496                                                                                ##STR42##                                                      35   FR140497                                                                                ##STR43##                                                      36   FR143483                                                                                ##STR44##                                                      37   FR140728                                                                                ##STR45##                                                      38   FR142172                                                                                ##STR46##                                                      39   FR143326                                                                                ##STR47##                                                      40   FR142390                                                                                ##STR48##                                                      41   FR140729                                                                                ##STR49##                                                      42   FR140730                                                                                ##STR50##                                                      43   FR143020                                                                                ##STR51##                                                      44   FR143021                                                                                ##STR52##                                                      45   FR141315                                                                                ##STR53##                                                      46   FR140105                                                                                ##STR54##                                                      47   FR141564                                                                                ##STR55##                                                      48   FR143170                                                                                ##STR56##                                                      49   FR138912                                                                                ##STR57##                                                      50   FR138960                                                                                ##STR58##                                                      51   FR138727                                                                                ##STR59##                                                      52   FR138912                                                                                ##STR60##                                                      53   FR138960                                                                                ##STR61##                                                      ______________________________________                                    

EXAMPLE 13

FR139835 substance was obtained by reacting FR133303 substance withN-octyloxycarbonyloxysuccinimide according to a method similar to thatof Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1137 (M+Na)

EXAMPLE 14

FR139537 substance was obtained by reacting FR133303 substance withsuccinimido 4-t-butylbenzoate according to a method similar to that ofExamDle 3.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (D₂ O, δ): 1.05 (3H, d, J=6.9 Hz), 1.15 (3H, d, J=5.9 Hz) , 1.33(9H, s), 2.0-2.3 (3H, m), 2.4-2.6 (3H, m), 2.7-2.9 (1H, m), 3.4-3.6 (1H,m), 3.8-4.9 (12H, m), 5.07 (2H, m), 5.40 (1H, d, J=3 Hz), 7.06 (1H, d,J=8.2 Hz), 7.08 (1H, dd, J=8.2 and 2 Hz), 7.27 (1H, d, J=2 Hz), 7.60(1H, d, J=8.6 Hz), 7.75 (1H, d, J=8.6 Hz )

EXAMPLE 15

FR141145 substance was obtained by reacting FR133303 substance withsuccinimido 4-(2-butoxyethoxy)benzoate according to a method similar tothat of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.88 (3H, t, J=7.3 Hz), 0.96 (3H, d, J=6.7 Hz),1.04 (3H, d, J=5.7 Hz), 1.2-1.6 (4H, m), 1.7-2.0 (3H, m), 2.1-2.65 (4H,m), 3.16 (1H, m), 3.7-4.5 (20H, m), 4.78 (1H, d, J=3 Hz), 4.86 (1H, d,J=3.8 Hz), 5.02 (1H, d, J=3 Hz), 6.74 (1H, d, J=8.2 Hz), 6.79 (1H, d,J=8.2 Hz), 7.00 (2H, d, J=8.9 Hz), 7.06 (1H, s), 7.87 (2H, d, J=8.9 Hz)FAB-MS e/z=1201 (M+Na)

EXAMPLE 16

FR139538 substance was obtained by reacting FR133303 substance withsuccinimido 4-(4-phenylbutoxy)benzoate according to a method similar tothat of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1233 (M+Na)

EXAMPLE 17

FR140215 substance was obtained by reacting FR133303 substance with4-octyloxyphthalic anhydride according to a method similar to that ofExample 3.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1257 (M+Na)

EXAMPLE 18

FR140216 substance was obtained by reacting FR133303 substance withsuccinimido 3-methoxy-4-octyloxybenzoate according to a method similarto that of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1243 (M+Na)

EXAMPLE 19

FR140727 substance was obtained by reacting FR133303 substance withsuccinimido4-(2,2,3,3,4,4,5,5-octafluoropentyloxy)-2,3,5,6-tetrafluorobenzoateaccording to a method similar to that of EXample 3.

IR (Nujol): 3300, 1630 cm⁻¹

FAB-MS e/z=1387 (M+Na)

EXAMPLE 20

FR143301 substance was obtained by reacting FR133303 substance withsuccinimido4-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyloxy)-2,3,5,6-tetrafluorobenzoateaccording to a method similar to that of Example 3.

IR (Nujol): 3300, 1630 cm⁻¹

FAB-MS e/z=1534 (M⁺)

EXAMPLE 21

FR140495 substance was obtained by reacting FR133303 substance withsuccinimido 2-(4-biphenylyl)acetate according to a method similar tothat of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 1.0-1.1 (6H, m), 1.9-2.2 (3H, m), 2.3-2.6 (3H, m),2.7-2.85 (1H, m), 3.35 (1H, M), 3.58 (2H, s), 3.65-4.7 (13H, m), 4.93(1H, d, J=3 Hz), 5.04 (1H, d, J=3.8 Hz), 5.25 (1H, d, J=3 Hz), 6.85 (1H,d, J=8.3 Hz), 7.01 (1H, dd, J=8.3 and 2 Hz ) , 7.3-7.6 (10H, m)

EXAMPLE 22

FR139503 substance was obtained by reacting FR133303 substance withsuccinimido 2-(4-octyloxyphenyl)-2-methoxyacetate according to a methodsimilar to that of Example 3.

IR (Nujol): 3330, 1620 cm⁻¹

FAB-MS e/z=1257 (M+Na)

EXAMPLE 23

FR139500 substance was obtained by reacting FR133303 substance with O⁴-octyl-N-(t-butoxycarbonyl)-D-tyrosine succinimido ester according to amethod similar to that of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.90 (3H, t, J=6.8 Hz), 1.06 (3H, d, J=6.8 Hz), 1.17(3H, d, J=6.7 Hz), 1.20-1.30 (10H, m), 1.35 (9H, s), 1.74 (2H, m),1.9-2.1 (3H, m), 2.45 (3H, m), 2.76 (1H, m), 3.0-3.1 (1H, m), 3.37 (1H,m), 3.7-4.6 (18H, m), 4.94 (1H, d, J=3 Hz), 5.01 (1H, d, J=3.8 Hz), 5.25(1H, d, J=3 Hz), 6.79 (2H, d, J=8.5 Hz), 6.83 (1H, d, J=8.3 Hz), 7.03(1H, dd, J=8.3 and 2 Hz), 7.12 (2H, d, J=8.5 Hz), 7.31 (1H, d, J=2 Hz)

EXAMPLE 24

FR139501 substance was obtained by reacting FR133303 substance withN-(t-butoxycarbonyl)-L-2-(2-naphthyl)glycine succinimido ester accordingto a method similar to that of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

EXAMPLE 25

FR139502 substance was obtained by reacting FR133303 substance withN.sup.τ -octyl-N-(t-butoxycarbonyl)-L-histidine succinimido esteraccording to a method similar to that of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1330 (M+Na)

EXAMPLE 26

FR138959 substance was obtained by reacting FR133303 substance withsuccinimido 2-(4-octyloxyphenyl)-2-methoxyiminoacetate according to amethod similar to that of Example 3.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.91 (3H, t, J=6.6 Hz), 1.06 (3H, d, J=6.8 Hz), 1.25(3H, d, J=6.3 Hz), 1.25-1.6 (10H, m), 1.65-1.9 (2H, m), 1.9-2.2 (3H, m),2.3-2.65 (3H, m), 1.75-1.9 (1H, m), 3.3-3.5 (1H, m), 3.95 (3H, s),3.7-4.75 (16H, m), 5.03 (1H, d, J=3.0 Hz), 5.11 (1H, d, J=3.7 Hz), 5.46(1H, d, J=2.7 Hz), 6.86 (1H, d, J=8.2 Hz), 6.89 (2H, d, J=8.9 Hz), 7.01(1H, dd, J=8.2 and 2 Hz), 7.31 (1H, d, J=2 Hz), 7.54 (2H, d, J=8.9 Hz)

FAB-MS e/z=1270 (M+Na)

EXAMPLE 27

FR140291 substance was obtained by reacting FR133303 substance withsuccinimido 2-(4-hydroxyphenyl)-2-(4-octyloxybenzyloxyimino)acetateaccording to a method similar to that of Example 3.

IR (Nujol): 3250, 1650, 1620 cm⁻¹

FAB-MS e/z=1363 (M+Na)

EXAMPLE 28

FR141580 substance was obtained by reacting FR133303 substance withsuccinimido 2-phenyl-2-(4-octyloxybenzyloxyimino)acetate according to amethod similar to that of Example 3.

IR (Nujol): 3300, 1646 cm⁻¹

FAB-MS e/z=1346 (M+Na)

EXAMPLE 29

FR141579 substance was obtained by reacting FR133303 substance withsuccinimido 2-(4-octyloxybenzyloxyimino)acetate according to a methodsimilar to that of Example 3.

IR (Nujol): 3250, 1650 cm⁻¹

FAB-MS e/z=1270 (M+Na)

EXAMPLE 30

FR141146 substance was obtained by reacting FR133303 substance with1-[(2E,6E)-3,7,11-trimethyl-2,6,10-dodecatrienoyl]-1H-benzotriazole-3-oxideaccording to a method similar to that of Example 12.

IR (Nujol): 3300, 1620, 1040 cm⁻¹

NMR (CD₃ OD, δ): 1.06 (3H, d, J.=6.8 Hz), 1.19 (3H, d, J=5.9 Hz), 1.60(3H, s), 1.62 (3H, s), 1.66 (3H, s), 1.9-2.2 (11H, m), 2.05 (3H, s),2.3-2.6 (3H, m), 2.7-2.9 (1H, m), 3.35 (1H, m), 3.7-5.0 (14H, m), 5.08(4H, m), 5.27 (1H, d, J=2.8 Hz), 5.77 (1H, s), 6.86 (1H, d, J=8.3 Hz),7.04 (1H, dd, J=8.3 and 1.9 Hz), 7.32 (1H, d, J=1.9 Hz)

EXAMPLE 31

FR140731 substance was obtained by reacting FR133303 substance with1-(4-octylbenzoyl)-1H-benzotriazole-3-oxide according to a methodsimilar to that of Example 12.

IR (Nujol): 3300, 1620, 1040 cm⁻¹

NMR (CD₃ OD, δ): 0.86 (3H, t, J=6.8 Hz), 1.06 (3H, d, J=6.8 Hz), 1.21(3H, d, J=5.8 Hz), 1.25-1.45 (10H, m), 1.55-1.75 (2H, m), 1.9-2.25 (3H,m), 2.35-2.6 (3H, m), 2.65 (2H, t, J=7.5 Hz), 2.81 (1H, m), 3.32 (1H,m), 3.7-4.8 (14H, m), 4.98 (1H, d, J=3 Hz), 5.09 (1H, d, J=3.9 Hz), 5.31(1H, d, J=3 Hz), 6.86 (1H, d, J=8.3 Hz), 7.03 (1H, dd, J=8.3 and 2 Hz),7.24 (2H, d, J=8.2 Hz), 7.33 (1H, d, J=2 Hz), 7.74 (2H, d, J=8.2 Hz)

FAB-MS e/z=1197 (M+Na)

EXAMPLE 32

FR140217 substance was obtained by reacting FR133303 substance with1-[4-(4-octyloxy)phenoxy]benzoyl-1H-benzotriazole-3-oxide according to amethod similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1305 (M+Na)

EXAMPLE 33

FR142472 substance was obtained by reacting FR133303 substance with1-[4-(4-octyloxyphenyl)benzoyl]-1H-benzotriazole-3-oxide according to amethod similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.88 (3H, t, J=6.7 Hz), 1.06 (3H, d, J=6.8 Hz), 1.23(3H, d, J=6.1 Hz), 1.3-1.6 (10H, m), 1.8-1.9 (2H, m), 1.9-2.3 (3H, m),2.3-2.7 (3H, m), 2.9-3.0 (1H, m), 3.39 (1H, m), 3.7-4.7 (16H, m), 4.99(1H, d, J=3.0 Hz), 5.10 (1H, d, J=3.7 Hz), 5.35 (1H, d, J=2.7 Hz), 6.87(1H, d, J=8.3 Hz), 6.99 (2H, d, J=8.8 Hz), 7.04 (1H, dd, J=8.3 and 1.9Hz), 7.33 (1H, d, J=1.9 Hz), 7.58 (2H, d, J=8.8 Hz), 7.62 (2H, d, J=8.4Hz), 7.87 (2H, d, J=8.4 Hz)

FAB-MS e/z=1289 (M+Na)

EXAMPLE 34

FR140496 substance was obtained by reacting FR133303 substance with1-(6-butoxy-2-naphthoyl)-1H-benzotriazole-3-oxide according to a methodsimilar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1207 (M+Na)

EXAMPLE 35

FR140497 substance was obtained by reacting FR133303 substance with1-(6-hexyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide according to amethod similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.89 (3H, t, J=6.6 Hz), 0.97 (3H, d, J=6.9 Hz),1.08 (3H, d, J=5.9 Hz), 1.2-1.6 (6H, m), 1.7-2.1 (5H, m), 2.1-2.5 (3H,m), 2.5-2.7 (1H, m), 3.19 (1H, m), 3.73 (2H, m), 3.8-4.5 (12H, m), 4.80(1H, d, J=3 Hz), 4.88 (1H, d, J=3.8 Hz), 5.08 (1H, d, J=3 Hz), 6.74 (1H,d, J=8.2 Hz), 6.80 (1H, dd, J=8.2 and 2 Hz), 7.08 (1H, d, J=2 Hz), 7.26(1H, dd, J=8.9 and 2.4 Hz), 7.39 (1H, d, J=2.4 Hz), 7.85 (1H, d, J=8.7Hz), 7.89 (1H, d, J=8.7 Hz), 7.93 (1H, d, J=8.9 Hz), 8.44 (1H, s)

FAB-MS e/z=1236 (M+Na)

EXAMPLE 36

FR143483 substance was obtained by reacting FR133303 substance with1-[6-(2-ethylhexyloxy)-2-naphthoyl]-1H-benzotriazole-3-oxide accordingto a method similar to that of Example 12.

IR (Nujol): 3250, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.93 (3H, t, J=7.4 Hz), 0.98 (3H, t, J=7.4 Hz), 1.06(3H, d, J=6.8 Hz), 1.24 (3H, d, J=6.0 Hz), 1.3-1.7 (8H, m), 1.7-1.9 (1H,m), 1.9-2.3 (3H, m), 2.3-2.7 (3H, m), 2.8-3.0 (1H, m), 3.39 (1H, m),3.7-4.7 (16H, m), 5.00 (1H, d, J=4.4 Hz), 5.11 (1H, d, J=3.7 Hz), 5.37(1H, d, J=2.6 Hz), 6.87 (1H, d, J=8.3 Hz), 7.04 (1H, dd, J=8.3 and 2 Hz), 7.17 (1H, dd, J=8.9 and 1.9 Hz), 7.22 (1H, d, J=2 Hz), 7.33 (1H, d,J=1.9 Hz), 7.7-7.9 (3H, m), 8.29 (1H, s)

FAB-MS e/z=1263 (M+Na)

EXAMPLE 37

FR140728 substance was obtained by reacting FR133303 substance with1-(6-decyloxy-2-naphthoyl) -1H-benzotriazole-3-oxide according to amethod similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.86 (3H, t, J=6.6 Hz), 0.97 (3H, d, J=6.7 Hz),1.07 (3H, d, J=5.9 Hz), 1.2-1.6 (14H, m), 1.7-2.1 (5H, m), 2.1-2.5 (3H,m), 2.5-2.7 (1H, m), 3.19 (1H, m), 3.45 (1H, m), 3.73 (2H, m), 3.9-4.5(12H, m), 4.79 (1H, d, J=3 Hz), 4.87 (1H, d, J=3.8 Hz), 5.07 (1H, d, J=3Hz), 6.74 (1H, d, J=8.2 Hz), 6.79 (1H, dd, J=8.1 and 2 Hz), 7.06 (1H, d,J=2 Hz ), 7.23 (1H, dd, J=8.9 and 2.4 Hz), 7.38 (1H, d, J=2.4 Hz), 7.85(1H, d, J=8.7 Hz), 7.89 (1H, d, J=8.7 Hz), 7.93 (1H, d, J=8.9 Hz), 8.45(1H, s)

FAB-MS e/z=1291 (M+Na)

EXAMPLE 38

FR142172 substance was obtained by reacting FR133303 substance with1-[6-(3,7-dimethyloctyloxy)-2-naphthoyl]-1H-benzotriazole-3-oxideaccording to a method similar to that of Example 12.

IR (Nujol): 3300, 1610 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.85 (6H, d, J=6.6 Hz), 0.95 (3H, d, J=5.9 Hz),0.97 (3H, d, J=6.7 Hz), 1.08 (3H, d, J=5.9 Hz), 1.1-1.4 (6H, m), 1.4-2.1(7H, m), 2.1-2.5 (3H, m), 2.5-2.7 (1H, m), 3.19 (1H, m), 3.74 (2H, m),3.9-4.6 (12H, m), 4.81 (1H, d, J=3 Hz), 4.87 (1H, d, J=3.8 Hz), 5.07(1H, d, J=3 Hz), 6.74 (1H, d, J=8.2 Hz), 6.83 (1H, dd, J=8.1 and 2 Hz),7.06 (1H, d, J=2 Hz), 7.23 (1H, dd, J=8.9 and 2.4 Hz), 7.40 (1H, d,J=2.4 Hz), 7.85 (1H, d, J=8.7 Hz), 7.89 (1H, d, J=8.7 Hz), 7.93 (1H, d,J=8.9 Hz), 8.45 (1H, s)

FAB-MS e/z=1291 (M+Na)

EXAMPLE 39

FR143326 substance was obtained by reacting FR133303 substance with1-[6-(3,7-dimethyl-6-octenyloxy) -2-naphthoyl]-1H-benzotriazole-3-oxideaccording to a method similar to that of Example 12.

IR (Nujol): 3300, 1620, 1260, 1040 cm⁻¹

NMR (CD₃ OD, δ): 1.00 (3H, d, J=6.2 Hz), 1.06 (3H, d, J=6.8 Hz), 1.25(3H, d, J=5.9 Hz), 1.2-1.6 (2H, m), 1.61 (3H, s), 1.67 (3H, s), 1.63-2.3(8H, m), 2.3-2.7 (3H, m), 2.8-3.0 (1H, m), 3.39 (1H, m), 3.7-4.8 (16H,m), 5.00 (1H, d, J=5.1 Hz), 5.08-5.2 (2H, m), 5.37 (1H, d, J=2.5 Hz),6.87 (1H, d, J=8.3 Hz), 7.04 (1H, d, J=8.3 Hz), 7.15 (1H, d, J=8.9 Hz),7.21 (1H, s), 7.33 (1H, s), 7.71 (1H, d, J=8.7 Hz), 7.77-7.85 (2H, m),8.28 (1H, s)

EXAMPLE 40

FR142390 substance was obtained by reacting FR133303 substance with1-[6-{(E)-3,7-dimethyl-2,6-octadienyloxy}-2-naphthoyl]-1H-benzotriazole-3-oxideaccording to a method similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.97 (3H, d, J=6.7 Hz), 1.07 (3H, d, J=6.0 Hz),1.57 (3H, s), 1.61 (3H, s), 1.76 (3H, s), 1.8-2.5 (9H, m), 2.5-2.7 (1H,m), 3.19 (1H, m), 3.45 (1H, m), 3.73 (2H, m), 3.9-4.6 (11H, m), 4.70(2H, d, J=6.5 Hz), 4.80 (1H, d, J=3 Hz), 4.87 (1H, d, J=3.8 Hz), 5.07(2H, m), 5.51 (1H, t, J=6.5 Hz), 6.74 (1H, d, J=8.3 Hz ), 6.83 (1H, dd,J=8.3 and 2 Hz), 7.07 (1H, d, J=2 Hz ), 7.24 (1H, dd, J=8.9 and 2.4 Hz), 7.40 (1H, d, J=2.4 Hz), 7.8-8.0 (3H, m), 8.45 (1H, s)

FAB-MS e/z=1287 (M+Na )

EXAMPLE 41

FR140729 substance was obtained by reacting FR133303 substance with1-(6-dodecyloxy-2-naphthoyl)-1H-benzotriazole-3-oxide according to amethod similar to that of Example 12.

IR (Nujol): 3300, 1610 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.85 (3H, t, J=6.6 Hz), 0.97 (3H, d, J=6.7 Hz),1.07 (3H, d, J=5.9 Hz), 1.2-1.6 (18H, m), 1.7-2.1 (5H, m), 2.1-2.5 (3H,m), 2.5-2.7 (1H, m), 3.19 (1H, m), 3.45 (1H, m), 3.73 (2H, m), 3.9-4.5(12H, m), 4.79 (1H, d, J=3 Hz), 4.87 (1H, d, J=3.8 Hz), 5.07 (1H, d, J=3Hz), 6.74 (1H, d, J=8.1 Hz), 6.78 (1H, dd, J=8.1 and 2 Hz), 7.06 (1H, d,J=2 Hz ), 7.23 (1H, dd, J=8.9 and 2.4 Hz), 7.38 (1H, d, J=2.4 Hz), 7.85(1H, d, J=8.7 Hz), 7.89 (1H, d, J=8.7 Hz), 7.93 (1H, d, J=8.9 Hz), 8.44(1H, s)

FAB-MS e/z=1320 (M+Na)

EXAMPLE 42

FR140730 substance was obtained by reacting FR133303 substance with1-(2-anthrylcarbonyl)-1H-benzotriazole-3-oxide according to a methodsimilar to that of Example 12

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1185 (M+Na)

EXAMPLE 43

FR143020 substance was obtained by reacting FR133303 substance with1-[2-(4-octyloxyphenyl)acetyl]-1H-benzotriazole-3-oxide according to amethod similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.87 (3H, t, J=6.8 Hz), 1.0-1.2 (6H, m), 1.2-1.6 (10H,m), 1.6-1.85 (2H, m), 1.85-2.1 (3H, m), 2.3-2.6 (3H, m), 2.7-2.85 (1H,m), 3.32 (1H, m), 3.46 (2H, s), 3.7-4.7 (16H, m), 5.04 (1H, d, J=3.7Hz), 5.23 (1H, d, J=2.7 Hz), 6.75-6.9 (3H, m), 7.01 (1H, d, J=8.3 Hz),7.15 (2H, d, J=8.5 Hz), 7.30 (1H, s)

FAB-MS e/z=1227 (M+Na)

EXAMPLE 44

FR143021 substance was obtained by reacting FR133303 substance with1-[3-(4-octyloxyphenyl)propionyl]-1H-benzotriazole-3-oxide according toa method similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1241 (M+Na)

EXAMPLE 45

FR141315 substance was obtained by reacting FR133303 substance with1-[(E)-3-(4-octyloxyphenyl)acryloyl]-1H-benzotriazole-3-oxide accordingto a method similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.86 (3H, t, J=6.7 Hz), 0.97 (3H, d, J=6.7 Hz),1.04 (3H, d, J=5.4 Hz), 1.2-1.5 (10H, m), 1.6-2.0 (5H, m), 2.1-2.5 (3H,m), 2.5-2.6 (1H, m), 3.17 (1H, m), 3.3-4.5 (15H, m), 4.79 (1H, d, J=3Hz), 4.86 (1H, d, J=3.8 Hz), 5.01 (1H, d, J=3 Hz), 6.57 (1H, d, J=15.8Hz), 6.74 (1H, d, J=8.2 Hz), 6.82 (1H, d, J=8.2 Hz), 6.97 (2H, d, J=8.8Hz), 7.09 (1H, s), 7.34 (1H, d, J=15.8 Hz), 7.52 (2H, d, J=8.8 Hz)

FAB-MS e/z=1239 (M+Na)

EXAMPLE 46

FR140105 substance was obtained by reacting FR133303 substance with1-(O⁴ -octyl-N,N-dimethyl-L-tyrosyl)-1-H-benzotriazole-3-oxide accordingto a method similar to that of Example 12.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.91 (3H, t, J=6.8 Hz), 1.06 (3H, d, J=6.8 Hz), 1.12(3H, d, J=6.1 Hz), 1.33 (10H, m), 1.74 (2H, m), 1.98 (3H, m), 2.40 (6H,s), 2.3-2.6 (3H, m), 2.8 (2H, m), 2.9-3.1 (1H, m), 3.3-3.5 (2H, m),3.6-4.7 (16H, m), 5.06 (1H, d, J=3.8 Hz), 5.33 (1H, d, J=3 Hz), 6.77(2H, d, J=8.6 Hz), 6.86 (1H, d, J=8.3 Hz), 7.03 (1H, dd, J=8.3 and 2Hz), 7.07 (2H, d, J=8.6 Hz), 7.31 (1H, d, J=2 Hz)

EXAMPLE 47

FR141564 substance was obtained by reacting FR133303 substance with4-octyloxyphenylsulfonyl chloride according to a method similar to thatof Example 6.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (DMSO-d₆ +D₂ O, δ): 0.87 (3H, t, J=6.7 Hz), 0.97 (3H, d, J=6.8 Hz),1.04 (3H, d, J=5.7 Hz), 1.1-1.5 (10H, m), 1.6-2.1 (5H, m), 2.45 (3H, m),2.5-2.7 (1H, m), 3.19 (1H, m), 3.7-4.5 (16H, m), 4.80 (1H, d, J=3 Hz),4.88 (1H, d, J=4 Hz), 5.08 (1H, d, J=3 Hz), 6.74 (1H, d, J=8.2 Hz), 6.82(1H, d, J=8.2 Hz), 6.84 (2H, d, J=8.7 Hz), 7.07 (1H, s), 7.51 (2H, d,J=8.7 Hz)

FAB-MS e/z=1249 (M+Na)

EXAMPLE 48

FR143170 substance was obtained by reacting FR133303 substance with6-octyloxy-2-naphthylsulfonyl chloride according to a method similar tothat of Example 6.

IR (Nujol): 3300, 1620 cm⁻¹

NMR (CD₃ OD, δ): 0.29 (3H, d, J=6.0 Hz), 0.91 (3H, t, J=6.7 Hz), 1.07(3H, d, J=6.9 Hz), 1.25-1.6 (10H, m), 1.7-2.2 (5H, m), 2.2-2.6 (4H, m),3.37 (1H, m), 3.55-4.65 (17H, m), 4.97 (1H, m), 5.54 (1H, m), 6.84 (1H,d, J=8.3 Hz), 7.01 (1H, dd, J=8.4 and 2 Hz), 7.15-7.3 (3H, m), 7.75-8.0(3H, m), 8.35 (1H, s)

FAB-MS e/z=1299 (M+Na)

EXAMPLE 49

To a solution of FR138364 substance obtained in Example 5 (0.24 g) inacetonitrile (5 ml), p-toluenesulfonic acid (0.132 g) was added, and themixture was stirred for 8 hours at room temperature. The reactionmixture was added to water and the aqueous layer was adjusted to pH 4.5with saturated aqueous sodium bicarbonate. The aqueous solution wassubjected to column chromatography on DIAION HP-20, eluting with 80%aqueous methanol. The fractions containing the object compound werecombined and evaporated under reduced pressure to remove methanol. Theresidue was lyophilized to give FR138912 substance (0.15 g).

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1272 (M+K)

EXAMPLE 50

A mixture of FR138728 substance obtained in Example 8 (0.15 g) and1-octyl-1,4-dihydropyridine-4-thione (0.031 g) in N,N-dimethylformamidewas stirred for 1.5 hours under ice-cooling. The reaction mixture waspulverized in diethyl ether (50 ml). The precipitate was filtered anddried under reduced pressure in the presence of phosphorus pentoxide.The powder was added to water (300 ml) and adjusted to pH 4.5. Theaqueous solution was subjected to column chromatography on DIAION HP-20(50 ml) and eluted with 80% aqueous methanol. The fractions containingthe object compound were combined and evaporated under reduced pressureto remove methanol. The residue was lyophilized to give FR138960substance (0.15 g).

IR (Nujol): 3300, 1620 cm⁻¹

FAB-MS e/z=1222 (Free M+Na)

The following compounds (Examples 51 to 53) were obtained according tomethods similar to that of Example 3.

EXAMPLE 51

FR138727 substance

NMR (CD₃ OD, δ): 0.90 (3H, t, J=6.8 Hz), 1.05 (3H, d, J=6.8 Hz),1.17-1.33 (13H, m), 1.6-1.8 (2H, m), 1.9-2.1 (3H, m), 2.50 (1H, m), 2.75(1H, dd, J=16 and 4 Hz), 3.40 (1H, m), 3.7-3.8 (1H, M), 3.98 (2H, t,J=6.2 Hz), 3.9-4.2 (5H, m), 4.3-4.5 (5H, m), 4.5-4.7 (3H, m), 4.97 (1H,d, J=3 Hz), 5.06 (1H, s), 5.20 (1H, d, J=3 Hz), 5.40 (1H, d, J=3 Hz),6.85 (1H, d, J=8.3 Hz), 6.95 (2H, d, J=8.5 Hz), 7.02 (1H, d, J=8.3 Hz),7.30 (1H, d, J=8.5 Hz), 7.44 (1H, s)

EXAMPLE 52

FR138912 substance

IR (Nujol): 3300, 1620 cm⁻¹

EXAMPLE 53

FR138960 substance

IR (Nujol): 3300, 1620 cm⁻¹

The following compounds (Preparations 94 and 95) were obtained accordingto methods similar to that of Preparation 5.

Preparation 94

Succinimido 4-(4-heptyloxyphenyl)benzoate

IR (Nujol): 1760, 1740, 1600 cm⁻¹

NMR (CDCl₃, δ): 0.87 (3H, t, J=6.8 Hz), 1.2-1.7 (8H, m), 1.7-1.9 (2H,m), 2.92 (4H, s), 4.01 (2H, t, J=6.5 Hz), 7.00 (2H, d, J=8.8 Hz), 7.58(2H, d, J=8.8 Hz), 7.69 (2H, d, J=8.5 Hz), 8.17 (2H, d, J=8.5 Hz)

Preparation 95

Succinimido 4-(4-hexyloxyphenoxy)benzoate

IR (Nujol): 1760, 1720, 1600 cm⁻¹

NMR (CDCl₃, δ): 0.92 (3H, t, J=6.8 Hz), 1.2-1.5 (6H, m), 1.7-1.9 (2H,m), 2.90 (4H, s), 3.96 (2H, t, J=6.5 Hz), 6.9-7.1 (6H, m), 8.07 (2H, d,J=9 Hz)

The structures of the compounds of Examples 54 and 55 are shownhereinbelow: ##STR62##

    ______________________________________                                        Example                                                                              Compound                                                               No     No.       R                                                            ______________________________________                                        54     FR144274                                                               55     FR144271                                                                                 ##STR63##                                                   ______________________________________                                    

The following compounds (Examples 54 and 55) were obtained according tomethods similar to that of Example 3.

EXAMPLE 54

FR144274

IR (Nujol): 3300, 1620 cm⁻¹

Anal. Calcd. for C₅₅ H₇₃ N₈ SO₂₂ Na.6H₂ O: C: 48.53, H: 6.29, N: 8.23,S: 2.35

Found C: 48.36, H: 6.34, N: 8.15, S: 2.30

FAB-MS e/z=1275 (M+Na)

EXAMPLE 55

FR144271

Anal. Calcd. for C₅₄ H₇₁ N₈ SO₂₃ Na.6H₂ O C: 47.57, H: 6.14, N: 8.22, S:2.35

Found C: 47.58, H: 6.05, N: 8.18, S: 2.27

FAB-MS e/z=1277 (M+Na)

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       XaaThrXaaXaaXaaXaa                                                            15                                                                            __________________________________________________________________________

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method for the prevention or the treatment ofPneumocystis carinii pneumonia, which comprises administering aneffective amount of a polypeptide compound of the formula (SEQ ID NO:1): ##STR64## wherein R¹ is hydrogen or an acyl group,R² is hydroxy oracyloxy group, R³ is hydroxy or hydroxysulfonyloxy, R⁴ is hydrogen orcarbamoyl, and R⁵ and R⁶ are each hydrogen or hydroxy, with the provisothat R⁵ is hydrogen when R⁶ is hydrogen,or a pharmaceutically acceptablesalt thereof to a human being or an animal in need thereof.